A cooperative study between the Suwannee River Water Management District (SRWMD) and the U.S. Geological Survey (USGS) is evaluating sources of nitrate in water from selected springs and zones in the Upper Floridan aquifer in the Suwannee River Basin. A multi-tracer approach, which consists of the analysis of water samples for naturally occurring chemical and isotopic indicators, is being used to better understand sources and chronology of nitrate contamination in the middle Suwannee River region. In July and August 1997, water samples were collected and analyzed from six springs and two wells for major ions, nutrients, and dissolved organic carbon. These water samples also were analyzed for environmental isotopes [18O/16O, D/H, 13C/12C, 15N/14N] to determine sources of water and nitrate. Chlorofluorocarbons (CCl3F, CCl2F2, and C2Cl3F3) and tritium (3H) were analyzed to assess the apparent ages (residence time) of springwaters and water from the Upper Floridan aquifer. Delta 15N-NO3 values in water from the six springs range from 3.94 per mil (Little River Springs) to 8.39 per mil (Lafayette Blue Spring). The range of values indicates that nitrate in the sampled springwaters most likely originates from a mixture of inorganic (fertilizers) and organic (animal wastes) sources, although the higher delta 15N-NO3 value for Lafayette Blue Spring indicates that an organic source of nitrogen is likely at this site. Water samples from the two wells sampled in Lafayette County have high delta 15N-NO3 values of 10.98 and 12.1 per mil, indicating the likelihood of an organic source of nitrate. These two wells are located near dairy and poultry farms, where leachate from animal wastes may contribute nitrate to ground water. Based on analysis of chlorofluorocarbons in ground water, the mean residence time of water in springs ranges from about 12 to 25 years. Chlorofluorocarbons-modeled recharge dates for water samples from the two shallow zones in the Upper Floridan aquifer range from 1985 to 1989.