The K channel currents are important modulators of smooth muscle membrane potential and excitability. We assessed whether voltage-gated K currents from human myometrium are regulated by placental steroid hormones during pregnancy and labor. Pregnant human myometrial cells were isolated from samples obtained at cesarean section. Primary cultured cells were treated with 100 nM 17β-estradiol, 1 μM progesterone, or both hormones in combination for 24 h. Acute effects of the two hormones were also determined. The K currents were recorded using the standard whole-cell, patch-clamp technique. Primary cultures possessed both delayed rectifier (I_KV) and A-like (I_KA) voltage-gated K currents. The 24-h 17β-estradiol treatment caused a hyperpolarizing shift in the steady-state inactivation of both I_KV and I_KA. Progesterone treatment also shifted the inactivation of I_KA and increased I_KV amplitude by 60%–110%. Conversely, the combined treatment had no effect on these currents. Neither 17β-estradiol (0.1–1 μM) nor progesterone (1–5 μM) had any effect on the K current when applied acutely. These results show that 17β-estradiol should inhibit myometrial K channel activity, whereas progesterone is likely to have the opposite effect. These results are consistent with the respective procontractile and proquiescence roles for 17β-estradiol and progesterone in human uterus during pregnancy.