We tested the hypothesis that previously demonstrated gender differences in acetylcholine-induced vascular relaxation could involve diverse Na⁺,K⁺-ATPase functions. We determined Na⁺,K⁺-ATPase by measuring arterial ouabain-sensitive ⁸⁶Rb uptake in response to acetylcholine (Ach). We found a significant increase of Na-pump activity only in aortic rings from female rats (control 206±11 vs 367±29 nmol ⁸⁶Rb/K /min x g wt tissue, P<0.01). Ovariectomy eliminated sex differences on the Na⁺,K⁺-ATPase function and chronic in vivo hormone replacement with 17 -estradiol restored acetylcholine effect on the Na⁺,K⁺-ATPase. Because Ach acts by enhancing production of NO, we examined if the NO-donor sodium nitroprusside (SNP) mimicked the action of Ach on Na⁺,K⁺-ATPase activity. SNP increased ouabain-sensitive ⁸⁶Rb uptake in denuded female arteries (control 123±7 vs 197±12 nmol ⁸⁶Rb/K /min x g wt tissue, P<0.05). Methylene blue (an inhibitor of guanylate cyclase) and KT-5823 (a cyclic GMP-dependent kinase inhibitor) blocked the stimulatory action of SNP. Exposure of female thoracic aorta to the Na-K pump inhibitor ouabain, significantly decreased SNP-induced and acetylcholine-mediated relaxation of aortic rings. At the molecular level, Western blot analysis of arterial tissue revealed significant sex differences in the relative abundance of catalytic isoforms of the Na⁺,K⁺-ATPase. Female-derived aortas exhibited a greater proportion of alpha 2 isoform (44%) compared to male-derived aortas. Furthermore, estradiol upregulated the expression of alpha 2 mRNA in male arterial explants. Our results demonstrate that enhancement of acetylcholine-induced relaxation observed in female rats may be in part explained by 1) NO-dependent increase Na⁺,K⁺-ATPase activity in female vascular tissue, and 2) greater abundance of alpha 2 isoform of Na⁺,K⁺-ATPase in females.