The goal of this study was to examine the status of the renal nitric oxide (NO) system by determining NO synthase (NOS) isoform activity and expression within the three regions of the kidney in 14-wk-old male and female spontaneously hypertensive rats (SHR). NOS activity, and NOS1 and NOS3 protein expressions and localization were comparable in the renal cortex and outer medulla of male and female SHR. In contrast, male SHR had significantly less NOS1 and NOS3 enzymatic activity (0 ± 5 and 53 ± 7 pmol•mg-1 •30 min-1, respectively) compared with female SHR (37 ± 16 and 172 ± 40 pmol•mg-1 •30 min-1, respectively). Lower levels of inner medullary NOS1 activity in male SHR were associated with less NOS1 protein expression [45 ± 7 relative densitometric units (RDU)] and fewer NOS1-positive cells in the renal inner medulla compared with female SHR (79 ± 12 RDU). Phosphorylation of NOS3 is an important determinant of NOS activity. Male SHR had significantly greater phosphorylation of NOS3 on threonine 495 in the renal cortex compared with females (0.25 ± 0.05 vs. 0.15 ± 0.06 RDU). NOS3 phosphorylation was comparable in males and females in the other regions of the kidney. cGMP levels were measured as an indirect index of NO production. cGMP levels were significantly lower in the renal cortex (0.08 ± 0.01 pmol/mg) and inner medulla (0.43 ± 0.02 pmol/mg) of male SHR compared with females (cortex: 0.14 ± 0.02 pmol/mg; inner medulla: 0.56 ± 0.02 pmol/mg). Our data suggest that the effect of the sex of the animal on NOS activity and expression is different in the three regions of the SHR kidney and supports the hypothesis that male SHR have lower NO bioavailability compared with females. Copyright © 2005 by the American Physiological Society.