Fifty paddlefish (Polyodon spathula) collected from two sites on the Ohio River and from one site on the Cumberland River, USA, were examined to determine gonad polychlorinated biphenyl (PCB) and chlordane concentrations, amounts of plasma sex steroids (testosterone and estradiol), hepatic microsomal ethoxyresorufin-O-deethylase (EROD) activity, and the presence of immunoreactive cytochrome P450 1A (CYP1A) protein. Percent hatch and liver, spleen, and kidney histology were also determined. Gonad PCB and chlordane concentrations were significantly higher in Ohio River paddlefish than in Cumberland River paddlefish. Gonad PCB and chlordane concentration and gonad percent lipid were correlated in Ohio River paddlefish. Five of 10 Ohio River egg samples exceeded the Food and Drug Administration's action limit for chlordane (0.30 μg/g). Polychlorinated biphenyl congener-specific analysis detected predominantly the tetra-, penta-, and hexachlorobiphenyls in paddlefish testes. Plasma testosterone levels were significantly lower in males collected from the upper Ohio River site than those collected from the lower part of the river. There was no measurable hepatic microsomal EROD activity in any of the 50 paddlefish collected from the three sites. Western blotting analysis confirmed that a rabbit antitrout CYP1A1 IgG antibody did not recognize a CYP1A protein in paddlefish liver microsomes. Percent hatch was not significantly different in eggs collected from the Cumberland (88–96%) and Ohio Rivers (90–95%). Histological analysis of liver, spleen, and kidney detected the presence of hepatic steatosis and hemosiderosis, splenic lymphoid cell depletion, and hyperplasia of interrenal and chromaffin tissues. Immunosuppression, hepatic metabolic disorders, and altered neuroendocrine function may be occurring in Ohio River paddlefish. Results presented here suggest that organochlorine exposure may be jeopardizing the long-term health of Ohio River paddlefish and that additional investigation of contaminant effects on immune system function and hormone levels in paddlefish is warranted.