Gender differences exist in a variety of cardiovascular disorders. Sex hormones have been shown to mediate pulmonary artery (PA) vasodilation. However, the effects of fluctuations in physiologic sex hormone levels due to gender and menstrual cycle on PA vasoreactivity have not been clearly established yet. We hypothesized that gender and menstrual cycle affect PA vasoconstriction under both normoxic and hypoxic conditions. Isometric force displacement was measured in isolated PA rings from proestrus females (PF), estrus and diestrus females (E/DF) and male (M) Sprague-Dawley rats. The vasoconstrictor response under normoxic conditions (organ bath bubbled with 95% O₂/5% CO₂) was measured after stimulation with 80 mmol/l of KCl and 1 micromol/l of phenylephrine. Hypoxia was generated by changing the gas to 95% N₂/5% CO₂. PA rings from PF demonstrated an attenuated vasoconstrictor response to KCl when compared to rings from E/DF (75.58+/-3.2% vs. 92.43+/-4.24%, p<0.01). Rings from M also exhibited attenuated KCl-induced vasoconstriction when compared to E/DF (79.34+/-3.2% vs. 92.43+/-4.24%, p<0.05). PA rings from PF exhibited an attenuated vasoconstrictor response to phenylephrine when compared to E/DF (59.61+/-2.98% vs. 70.03+/-4.61%, p<0.05). While the maximum PA vasodilation during hypoxia did not differ between PF, E/DF and M, phase II of hypoxic pulmonary vasoconstriction was markedly diminished in the PA from PF (64.10+/-7.10% vs. 83.91+/-5.97% in M, p<0.05). We conclude that gender and menstrual cycle affect pulmonary artery vasoconstriction in isolated pulmonary artery rings. Even physiologic increases in circulating estrogen levels attenuate pulmonary artery vasoconstriction under both normoxic and hypoxic conditions.