Human health and ecological concerns have arisen due to a steady increase in nitrate-N concentrations during the past 40 years in Fannin Springs (0.3–4.7 mg/L), a regional discharge point with an average flow of >2.8 m3/second (>100 ft3/second) for water from the karstic Upper Floridan aquifer (UFA). Multiple chemical indicators (major dissolved species, 15N and 18O of nitrate, dissolved gases, 78 pesticides and degradates, and 67 organic compounds typically found in domestic and industrial wastewater) and transient tracers (3H/3He, chlorofluorocarbons [CFCs], sulfur hexafluoride [SF6]) were analyzed in water samples from nine wells along three transects and in spring water to assess groundwater age and potential contaminant sources. Land use is predominantly agricultural (52 percent) and forest (31 percent) in the 320 km2 (124 mi2) spring basin, which was delineated from a potentiometric-surface map of the UFA using high-resolution water-level data. Nitrate-N concentrations were highly variable in the oxic UFA and ranged from <0.02 to 4.7 mg/L. δ15N-NO3 values (3.4–9.9 per mil) indicated that nitrate contamination originated from inorganic sources (synthetic fertilizer) and organic sources (manure spreading or waste disposal). Higher nitrate concentrations and the younger age of spring water relative to water from upgradient wells indicate better communication with N sources at the surface. Apparent ages of groundwater correlated positively with well depth (P < 0.05) and were younger in water from wells nearer to the spring (<8 years) compared with other wells (10–50 years). Most transient tracer concentrations were consistent with binary mixing curves representing mixtures of water recharged during the past 10 years and older water (recharged before 1940). Young water mixing fractions ranged from 0.07 to 0.90. Trace levels of herbicides found in groundwater and spring water were indicative of applications for vegetative control in agricultural and other land-use types.