SummarySpecific arginine vasopressin (AVP) binding sites were identified and characterized using Leydig cell membranes prepared from a clonal murine Leydig‐derived cell line, TM3.3H‐AVP binding data analyses demonstrated that the radioligand binds to a high affinity, low capacity, homogeneous class of sites with a dissociation constant of 0.5 nM. Characterization of these AVP binding sites included competition studies. Displacement of3H‐AVP binding with high affinity by unlabelled AVP, LVP and the V1 antagonist, d(CH2)sTyr(Me)AVP, indicated that the Leydig cell AVP receptor is of the V1 type. Furthermore, AVP did not increase adenylate cyclase activity in TM3membranes, a finding consistent with the V1 type of AVP receptor. No competition with3H‐AVP was found with the V2 agonist, dVDAVP, or the selective oxytocin agonist, [Thr4, Gly7]oxytocin. No specific binding for oxytocin was found in Leydig cell membranes. No specific binding for either3H‐AVP or3H‐oxytocin was observed in membranes prepared from the Sertoli cell line or peritubular cell line. These findings indicate that murine Leydig cells have specific AVP binding sites of the V1 type. These AVP sites are not coupled to the adenylate cyc