The present study was undertaken to determine whether sex hormones influence nitric oxide synthase levels in the kidney. Five groups of rats were studied: males, castrated males, females, oophorectomized females, and oophorectomized females receiving estradiol replacement therapy. Endothelial nitric oxide synthase (eNOS) levels in the kidney were measured by Western blotting. eNOS levels were significantly greater in the renal medulla of female rats compared with male rats (3545 +/- 473 versus 2418 +/- 205 densitometry units (DU), P < 0.05). Oophorectomy reduced renal medullary eNOS levels to that of intact male rats (2566 +/- 304 DU, P = NS). Estrogen replacement therapy significantly increased medullary eNOS levels in oophorectomized animals (3249 +/- 377 versus 2302 +/- 213 DU, P < 0.05). Renal inducible nitric oxide synthase (iNOS) levels were measured after induction with lipopolysaccharide. iNOS levels were significantly greater in the renal medulla of female rats compared with male rats (677 +/- 253 versus 252 +/- 12 DU, P < 0.05). Oophorectomy reduced renal medullary iNOS levels to that of intact male rats (295 +/- 57 DU, P = NS). In contrast, estrogen replacement therapy significantly increased medullary iNOS levels in oophorectomized animals (682 +/- 356 versus 160 +/- 92 DU, P < 0.05). Steady-state levels of mRNA for iNOS were found to be higher in the inner medulla of female rats compared with male rats (1519 +/- 211 versus 899 +/- 105 DU, P < 0.05). In contrast to these findings, sex hormones failed to influence nitric oxide production or iNOS levels in lipopolysaccharide-stimulated mesangial cells in culture. These results suggest that gender may influence renal medullary synthesis of nitric oxide.