Previous work has shown that the genes encoding each α₁-adrenoceptor subtype are coexpressed throughout the peripheral vascular system. We have evaluated subtype-selective antibodies as tools to determine the extent of protein expression in arteries. The α_1A-, α_1B-, and α_1D-adrenoceptors were detected in the medial layer of the aorta, caudal, femoral, iliac, renal, superior mesenteric, and mesenteric resistance arteries. In Rat1 fibroblasts expressing each subtype, immunoreactivity was noted both on the cell surface and in a perinuclear orientation. Intense α_1B-adrenoceptor immunostaining was similarly localized in cultured femoral and renal vascular smooth muscle cells. Although the cellular localization appeared to be the same, immunoreactivity obtained with α_1A- and α_1D-adrenoceptors was much less intense than that with the α_1B-adrenoceptor. The α_1A-adrenoceptor selective agonist A-61603 was 22-fold more potent in activating renal artery contraction when compared with the femoral artery. The expression of each α₁-adrenoceptor was significantly decreased by in vivo application of antisense oligonucleotides targeted against each subtype. Inhibition of the expression of only one, the α_1A in renal and the α_1D in femoral arteries, reduced the contractile response to naphazoline. The results show: 1) subtype-selective antibodies can be used in tissues and cell culture to localize the α₁-adrenoceptor subtypes, 2) in addition to expression on the cell surface, the α₁-adrenoceptors are expressed intracellularly, and 3) despite expression of all adrenoceptors, a single subtype mediates the contractile response in the femoral and renal arteries.