The U.S. Geological Survey conducted a 4-year investigation of water quality and aquatic-community structure in Threemile Creek, an urban stream that drains residential areas in Mobile, Alabama. Water-quality samples were collected between March 2000 and September 2003 at four sites on Threemile Creek, and between March 2000 and October 2001 at two tributary sites that drain heavily urbanized areas in the watershed. Stream samples were analyzed for major ions, nutrients, fecal-indicator bacteria, and selected organic wastewater compounds. Continuous measurements of dissolved-oxygen concentrations, water temperature, specific conductance, and turbidity were recorded at three sites on Threemile Creek during 1999?2003. Aquatic-community structure was evaluated by conducting one survey of the benthic invertebrate community and multiple surveys of the algal community (periphyton). Benthic invertebrate samples were collected in July 2000 at four sites on Threemile Creek; periphyton samples were collected at four sites on Threemile Creek and the two tributary sites during 2000 ?2003. The occurrence and distribution of chemical constituents in the water column provided an initial assessment of water quality in the streams; the structure of the benthic invertebrate and algal communities provided an indication of the cumulative effects of water quality on the aquatic biota. Information contained in this report can be used by planners and resource managers in the evaluation of proposed total maximum daily loads and other restoration efforts that may be implemented on Threemile Creek. The three most upstream sites on Threemile Creek had similar water chemistry, characterized by a strong calcium-bicarbonate component; the most downstream site on Threemile Creek was affected by tidal fluctuations and mixing from Mobile Bay and had a strong sodium-chloride component. The water chemistry at the tributary site on Center Street was characterized by a strong sodium-chloride component; the water chemistry at the second tributary site, Toulmins Spring Branch, was characterized by a strong calcium component without a dominant anionic species. The ratios of sodium to chloride at the tributary at Center Street were higher than typical values for seawater, indicating that sources other than seawater (such as leaking or overflowing sewer systems or industrial discharge) likely are contributors to the increased levels of sodium and chloride. Concentrations of fluoride and boron also were elevated at this site, indicating possible anthropogenic sources. Dissolved-oxygen concentrations were not always within levels established by the Alabama Department of Environmental Management; continuous monitors recorded dissolved-oxygen concentrations that were repeatedly less than the minimum criterion (3.0 milligrams per liter) at the most downstream site on Threemile Creek. Water temperature exceeded the recommended criterion (32.2 degrees Celsius) at five of six sites in the Threemile Creek basin. The pH values were within established criteria (6.0 ? 8.5) at sites on Threemile Creek; however, pH values ranged from 7.2 to 10.0 at the tributary at Center Street and from 6.6 to 9.9 at Toulmins Spring Branch. Nutrient concentrations in the Threemile Creek basin reflect the influences of both land use and the complex hydrologic systems in the lower part of the basin. Nitrite-plus-nitrate concentrations exceeded U.S. Environmental Protection Agency ecoregion nutrient criteria in 88 percent of the samples. In 45 percent of the samples, total phosphorus concentrations exceeded the U.S. Environmental Protection Agency goal of 0.1 milligram per liter for preventing nuisance aquatic growth. Ratios of nitrogen to phosphorus indicate that both nutrients have limiting effects. Median concentrations of enterococci and fecal coliform bacteria were highest at the two tributary sites and lowest at the most upstream site on Threemile Creek. In general, concentrations o