Multiple chemical constituents (nutrients; N, O, H, C stable isotopes; 64 organic wastewater compounds, 16 pharmaceutical compounds) and microbiological indicators were used to assess the impact on groundwater quality from the land application of approximately 9.5 million liters per day of treated municipal sewage effluent to a sprayfield in the 960-km2 Ichetucknee Springs basin, northern Florida. Enriched stable isotope signatures (?? 18O and ??2H) were found in water from the effluent reservoir and a sprayfield monitoring well (MW-7) due to evaporation; however, groundwater samples downgradient from the sprayfield have ??18O and ??2H concentrations that represented recharge of meteoric water. Boron and chloride concentrations also were elevated in water from the sprayfield effluent reservoir and MW-7, but concentrations in groundwater decreased substantially with distance downgradient to background levels in the springs (about 12 km) and indicated at least a tenfold dilution factor. Nitrate-nitrogen isotope (??15N-NO3) values above 10 ??? in most water samples were indicative of organic nitrogen sources except Blue Hole Spring (??15N-NO3 = 4.6-4.9 ???), which indicated an inorganic source of nitrogen (fertilizers). The detection of low concentrations the insect repellent N,N-diethyl-metatoluamide (DEET), and other organic compounds associated with domestic wastewater in Devil's Eye Spring indicated that leakage from a nearby septic tank drainfield likely has occurred. Elevated levels of fecal coliforms and enterococci were found in Blue Hole Spring during higher flow conditions, which likely resulted from hydraulic connections to upgradient sinkholes and are consistent with previoius dye-trace studies. Enteroviruses were not detected in the sprayfield effluent reservoir, but were found in low concentrations in water samples from a downgradient well and Blue Hole Spring during high-flow conditions indicating a human wastewater source. The Upper Floridan aquifer in the Ichetucknee Springs basin is highly vulnerable to contamination from multiple anthropogenic sources throughout the springs basin. ?? 2007 Springer-Verlag.