The goals of this study were to (1) measure plasma osmolytes and rectal gland weights of a freshwater (FW) Atlantic stingray (Dasyatis sabina) population in the St. Johns River, Florida, and (2) determine how these parameters change after acclimation to seawater (SW). We hypothesized that the FW D. sabina may show physiological divergence from marine D. sabina, because the FW individuals reproduce and complete their life cycle in the St. Johns River. The FW D. sabina hyperregulate their plasma osmolality (621.4 mOsm kg(-1)), with plasma Na+, Cl-, and urea concentrations of 211.9, 207.8, and 195.9 mmol L(-1), respectively. FW D. sabina were exposed to 100% SW for 8 d, and their hematocrit did not change significantly compared to control animals left in FW. However, plasma osmolality increased significantly (953 mOsm kg(-1)), with significant increases in plasma concentrations of Na+, Cl-, and urea to 319.13, 296.1, and 329.76 mmol L(-1), respectively. The plasma of the SW-adapted D. sabina was hypo-osmotic and hypoionic to 100% SW. Rectal gland weight to body weight (RGBW) ratios of FW D. sabina were about 80% lower than RGBW ratios reported for marine D. sabina; the RGBW ratio did not increase significantly after SW acclimation. This may indicate that branchial and renal mechanisms are also involved with ion excretion. We conclude that the FW D. sabina are physiologically euryhaline and have not evolved the osmoregulatory strategy of stenohaline FW Potamotrygonid stingrays.