We have previously shown that chronic estrogen therapy improves endothelium-dependent vasodilation in the resistance vessels of biological males. Whether this is nitric oxide (NO) mediated and whether estrogen improves metabolic vasodilation is unknown. Resting forearm blood flow (FBF), ACh-induced vasodilation, and functional hyperemic blood flow (exercise) were assessed before and after the inhibition of NO with N(G)-monomethyl-L-arginine (L-NMMA) in 15 male-to-female transsexuals prescribed estrogen and in 14 age-matched males. Resting FBF was similar in the two groups and was similarly (P = 0.44) but significantly reduced by 48% after infusion of L-NMMA (P < 0.0001). The ACh dose-response relationship was shifted upward and to the left in the transsexual compared with the male group (P < 0.01). After the inhibition of NO, however, the difference in the ACh dose-response curve between the two groups was abolished (P = 0.15). Peak functional hyperemic blood flow was similar for the two groups (P = 0.94). L-NMMA produced a significant (P < 0.01) but similar (P = 0.64) reduction in peak hyperemia in the two groups. The volume of blood repaid to the forearm 1 and 5 min after exercise was also reduced by L-NMMA (P < 0.0001); however, there were no differences between the two groups. This suggests that ACh-mediated NO-dependent vasodilation may be more sensitive to the effects of chronic estrogen than exercise-induced vasodilation. Long-term estrogen does not appear to improve exercise-induced metabolic vasodilation in biological males, despite the fact that NO contributes to this process.