Uterine vascular tone significantly decreases whereas uterine blood flow dramatically increases during pregnancy. However, the complete molecular mechanisms remain elusive. We hypothesized that increased Ca2+-activated K+ (BKCa) channel activity contributes to the decreased myogenic tone of uterine arteries in pregnancy. Resistance-sized uterine arteries were isolated from nonpregnant and near-term pregnant sheep. Electrophysiological studies revealed a greater whole-cell K+ current density in pregnant compared with nonpregnant uterine arteries. Tetraethylammonium and iberiotoxin inhibited K+ currents to the same extent in uterine arterial myocytes. The BKCa channel current density was significantly increased in pregnant uterine arteries. In accordance, tetraethylammonium significantly increased pressure-induced myogenic tone in pregnant uterine arteries and abolished the difference in myogenic responses between pregnant and nonpregnant uterine arteries. Activation of protein kinase C produced a similar effect to tetraethylammonium by inhibiting BKCa channel activity and increasing myogenic tone in pregnant uterine arteries. Chronic treatment of nonpregnant uterine arteries with physiologically relevant concentrations of 17β-estradiol and progesterone caused a significant increase in the BKCa channel current density. Western blot analyses demonstrated a significant increase of the β1, but not α, subunit of BKCa channels in pregnant uterine arteries. In accordance, steroid treatment of nonpregnant uterine arteries resulted in an upregulation of the β1, but not α, subunit expression. The results indicate that the steroid hormone-mediated upregulation of the β1 subunit and BKCa channel activity may play a key role in attenuating myogenic tone of the uterine artery in pregnancy.