The ligand specificities of sites binding tritium-labeled arginine vasopressin (3H-AVP) were investigated in membrane preparations of rat anterior pituitary gland and liver. Labeled AVP interacted with high-affinity, low-capacity binding sites in liver as well as in pituitary membrane fractions. Binding displacement studies showed that AVP was a potent ligand for liver (K1 = 0.23 nM) and pituitary (K1 = 0.4 nM) receptors. Oxytocin and the antidiuretic (V2) agonist dDAVP had low affinities (K1 > 10 nM) both for liver and pituitary binding sites. The pressor (V1) antagonist d(CH2)5Tyr(Me)AVP was equipotent with AVP in liver membranes, but was 1,000-fold less potent on pituitary receptors. Another V1 antagonist, dPenTyr(Me)AVP, displaced 3H-AVP with K1 values of 3 and 8 nM from pituitary and liver receptors, respectively. These data are in reasonable agreement with recent studies of the in vitro biological activities of the analogs tested, and suggest that the ligand specificity of rat pituitary AVP receptors is distinct from those of previously characterized V1, V2 and oxytocin binding sites in the rat.