Ethanol inhibition of large-conductance, Ca²⁺-activated K⁺ (BK_Ca) channels in aortic myocytes may contribute to the direct contraction of aortic smooth muscle produced by acute alcohol exposure. In this tissue, BK_Ca channels consist of pore-forming (bslo) and modulatory () subunits. Here, modulation of aortic myocyte BK_Ca channels by acute alcohol was explored by expressing bslo subunits in Xenopus oocytes, in the absence and presence of ₁-subunits, and studying channel responses to clinically relevant concentrations of ethanol in excised membrane patches. Overall, average values of bslo channel activity (NP_o, with N = no. of channels present in the patch; P_o = probability of a single channel being open) in response to ethanol (3-200 mM) mildly decrease when compared with pre-ethanol, isosmotic controls. However, channel responses show qualitative heterogeneity at all ethanol concentrations. In the majority of patches (42/71 patches, i.e., 59%), a reversible reduction in NP_o is observed. In this subset, the maximal effect is obtained with 100 mM ethanol, at which NP_o reaches 46.2 ± 9% of control. The presence of ₁-subunits, which determines channel sensitivity to dihydrosoyaponin-I and 17-estradiol, fails to modify ethanol action on bslo channels. Ethanol inhibition of bslo channels results from a marked increase in the mean closed time. Although the voltage dependence of gating remains unaffected, the apparent effectiveness of Ca²⁺ to gate the channel is decreased by ethanol. These changes occur without modifications of channel conduction. In conclusion, a new molecular mechanism that may contribute to ethanol-induced aortic smooth muscle contraction has been identified and characterized: a functional interaction between ethanol and the bslo subunit and/or its lipid microenvironment, which leads to a decrease in BK_Ca channel activity.