Cultured vascular smooth muscle cells derived by enzymatic dissociation of rabbit aortic media were used for study of α1-adrenergic receptors (AAR) and receptor-coupled calcium flux. AAR were characterized by binding of the α1-selective radioligand [3H]prazosin. and norepinephrine-stimulated 45calcium efflux was measured as an index of AAR-mediated calcium flux. The binding of [3H]prazosin to a crude cellular homogenate was to a single, saturable site of high affinity (Kd 0.15 nM, Bmax = 75–125 fmol/mg protein), which was stereo-specific and exhibited the appropriate potency order for competition by agonists. Prazosin (Kd = 0.07 nM) was approximately 3000-fold more potent than the α2-selective antagonist yohimbine (Kd = 222 nM), both of which exhibited Hill coefficients of one. indicative of binding to a single receptor type. In intact cells, norepinephrine caused a concentration-related (EC50 - 100 nM) increase in 45calcium efflux which was blocked by low concentrations of prazosin (IC50 0.1 nM), but not yohimbine (IC50 (100 nM). An α1-selective concentration of prazosin (100 nM) fully blocked norepinephrine-stimulated 45calcium efflux, and therefore, it appeals that this effect does not involve α2-adrenergic receptors. Treatment of cultured cells with 1-norepinephrine for 48 h caused a concentration-related decrease in both AAR density and maximum norepinephrine-stimulated 45calcium efflux. This cultured vascular smooth muscle cell system provides several advantages for the study of α-adrenergic receptor coupling and regulation.