Daily estradiol-17 (E₂) increases basal uterine blood flow (UBF) and enhances acute E₂-mediated increases in UBF in ovariectomized nonpregnant ewes. The acute E₂-mediated rise in UBF involves vascular smooth muscle (VSM) Ca²⁺-activated K⁺ channels (BK_Ca). BK_Ca consist of pore forming -subunits and regulatory ₁-subunits that modulate channel function and E2 responsiveness. It is unclear if E₂ also alters subunit expression and thus channel density and/or function, thereby contributing to the rise in basal UBF and enhanced UBF responses that follow daily E₂. Therefore, we examined BK_Ca subunit expression using RT-PCR and immunoblot analysis of arterial VSM from reproductive and nonreproductive tissues and myometrium from ovariectomized nonpregnant ewes after daily E₂ (1 µg/kg iv) or vehicle without or with acute E₂ (1 µg/kg). Tissue distribution was determined by immunohistochemistry. Acute E₂ did not alter - or ₁-subunit expression in any tissue, P>0.1. Daily E₂ also did not affect -subunit mRNA or protein in any tissue (P>0.1) or mesenteric arterial VSM ₁-subunit. However, daily E₂ increased uterine and mammary arterial VSM ₁-subunit mRNA 32% and 83% (P<0.05), uterine VSM protein 30%, and myometrial ₁-subunit mRNA and protein 74% (P0.005). Immunostaining of uterine arteries, myometrium and intramyometrial arteries paralleled immunoblot analyses for both subunits. Although BKCa density is unaffected by daily and acute E₂, daily E₂ increases ₁-subunit in proximal and distal uterine arterial VSM. Thus prolonged E₂ exposure may alter BK_Ca function, estrogen responsiveness, and basal vascular tone and reactivity in reproductive arteries by modifying :₁ stoichiometry.