We investigated the binding of 3H-Arg8-vasopressin to membranes from rat mesenteric arteries. Specific binding of 3H-vasopressin was 60-75% of total binding. Binding at 22 C achieved a plateau at 30 min whereas at 4 C binding was significantly slower. Binding was reversible upon addition of 1 microM Arg8-vasopressin after 30 min of incubation. Scatchard analysis indicated a single class of high-affinity binding sites with an equilibrium dissociation constant of 5.1 +/- 0.6 nM and a total binding capacity of 91 +/- 12 fmol/mg protein. Competitive inhibition of 3H-Arg8-vasopressin binding showed an IC50 of 3 nM for Arg8-vasopressin, 14 nM for [I-(beta-mercapto-beta-beta-cyclopentamethylene-propionic, 4-valine, 8-D-arginine]-vasopressin, 31 nM for oxytocin, 52 nM for I-deamino-8-D-arginine-vasopressin, 0.1 microM for [I-deamino-penicillamine, 4-valine, 8-D-arginine]-vasopressin, and 0.8 microM for desglycinamide-deamino-Arg8-vasopressin. Unrelated peptides did not displace 3H-Arg8-vasopressin. We conclude that these binding sites possess characteristics of physiologically relevant vasopressin receptors in vascular smooth muscle of a resistance type vessel.