In situ hybridization histochemistry, radioligand binding, and in vitro contractile studies were used to characterize the vascular distribution of the recently discovered α1D-adrenoceptor. In situ hybridization with an antisense probe localized the mRNA for the α1D-adrenoceptor to the medial layer of the rat aorta renal and mesenteric resistance arteries. If the tissues were first treated with RNase or a sense probe was used, no specific hybridization signal was detected. The extent to which this receptor was expressed as protein was assessed with radioligand binding studies. A series of ligands used to characterize α1-adrenoceptors interacted with two sites labeled by [3H]prazosin in homogenates from the aorta and mesenteric vascular bed. The high affinity site had the characteristics of an α1A-adrenoceptor. However, the low affinity site had ligand binding characteristics distinct from those of the α1D-adrenoceptor or any other known α1-adrenoceptor subtype. mRNA for the α1B-and α1C-adrenoceptors was also detected in the aorta, renal arteries, and mesenteric resistance arteries. Chloroethylclonidine (CEC) (1 and 10 μM) had differential effects on phenylephrine-induced contractions of vascular smooth muscle. CEC completely inhibited the response in the aorta and caused a partial inhibition in the mesenteric resistance artery. The same concentrations of CEC had little effect on phenylephrine responses in the renal artery. The data suggest the following. 1) mRNA for the novel α1D-adrenoceptor is localized in vascular smooth muscle. 2) Definitive identification of expression of this receptor was not possible; this may be related to coexpression of other subtypes of α1-adrenoceptors. 3) mRNA for the α1C-adrenoceptor was detected in rat peripheral vasculature. 4) The α1A-adrenoceptor is also localized in the vasculature. 5) Three and possibly four α1-adrenoceptors participate in vascular smooth muscle regulation.