Hydrographic and nutrient structure and the nitrogen and oxygen isotopic compositions (d15N and d18O) of the nitrate pool seaward of the Florida Keys reef tract were measured in Summer 2003. Nitrate and soluble reactive phosphate concentrations were tightly associated with depth and temperature and were close to 0 in surface waters, increased to 2–10 µmol L−1 and 0.1–1.0 µmol L−1, respectively, at intermediate depths associated with the thermocline and 15–25 µmol L−1 and 1.0–1.5 µmol L−1, respectively, in deeper water. Ammonium concentrations were low (0–0.6 µmol L−1) at all depths. Dissolved organic carbon concentrations ranged from 70–120 µmol L−1 near the surface to 40–60 µmol L−1 below the thermocline. Mean nitrate δ15N was 4.78% (±0.05) overall and varied from 4.24%(±0.07) within the chlorophyll maximum layer at 50–70 m depth to 5.26% (±0.05) below the thermocline at 150–242 m depth. Mean δ18O mean was 3.26% (±0.08) and varied from 4.89% (±0.14) within the chlorophyll maximum layer to 3.02% (±0.07) below the thermocline. These observations suggest an oceanic, deep-water nitrate source below the thermocline, a mix of sources from upwelling and N2 fixation, and the effects of fractionation associated with uptake by primary producers within the thermocline. The water column nitrate isotopic data support the general hypothesis that primary producers on the Florida Keys reef tract directly utilize the transiently available subsurface nitrate pool.