BACKGROUND AND PURPOSE: Ethanol at concentrations obtained in the circulation during moderate-heavy episodic drinking (30-60 mM) causes cerebral artery constriction in several species, including humans. In rodents, ethanol-induced cerebral artery constriction results from ethanol inhibition of large conductance voltage/Ca(2+)(i) -gated K(+) (BK) channels in cerebral artery myocytes. Moreover, the smooth muscle-abundant BK β1 accessory subunit is required for ethanol to inhibit cerebral artery myocyte BK channels under physiological Ca(2+)(i) and voltages and thus constrict cerebral arteries. The molecular bases of these ethanol actions remain unknown. Here, we set to identify the BK β1 region(s) that mediates ethanol-induced inhibition of cerebral artery myocyte BK channels and eventual arterial constriction. EXPERIMENTAL APPROACH: We used protein biochemistry, patch-clamp on engineered channel subunits, reversible cDNA permeabilization of KCNMB1 K/O mouse arteries and artery in vitro pressurization. KEY RESULTS: Ethanol inhibition of BK current was facilitated by β1 but not β4 subunits. Furthermore, only BK complexes containing β chimeras with β1 transmembrane (TM) domains on a β4 background or with a β1 TM2 domain on a β4 background displayed ethanol responses identical to those of BK complexes including wild-type β1. Moreover, β1 TM2 itself but not other β regions were necessary for ethanol-induced cerebral artery constriction. CONCLUSIONS AND IMPLICATIONS: BK β1 TM2 is necessary for this subunit to enable ethanol-induced inhibition of myocyte BK channels and cerebral artery constriction at physiological Ca(2+) and voltages. Thus, novel agents that target β1 TM2 may be considered to counteract ethanol-induced cerebral artery constriction and associated cerebrovascular conditions.