Abstract In this study oxytocin (OT) receptors have been characterized and localized in the testis of the rat using the radioiodinated OT receptor antagonist ¹²⁵I-labelled d(CH₂)⁵ [Tyr(Me)²,Thr⁴,Tyr⁹-NH₂]-vasotocin (OTA). Receptor density and localization have been compared with the rat testis arginine vasopressin (AVP) receptor using the radioiodinated AVP V_1a receptor antagonist ¹²⁵I-labelled d(CH₂)₅Sar⁷-AVP and the radioiodinated linear AVP V_1a antagonist ¹²⁵I-labelled [(C₆H₅-CH₂CO)-O-methyl-d-Tyr-Phe-Gln-Asn-Arg-Pro-Arg-Tyr-NH₂]. ¹²⁵I-labelled OTA bound with high affinity to membrane fractions of the rat testis (K_a = 13·8 ± 1·25 litres/nmol), mammary tissue (K_a=20·3± 4·36 litres/nmol) and uterus (K_a=27·8±0·74 litres/nmol). Competition studies with various OT and AVP receptor agonists and antagonists confirmed that the binding was to OT receptors. AVP receptors in the testis were found to be identical to AVP V_1a receptors in the liver. The AVP receptor density in the testis was much higher than the OT receptor density (109 ±12·3 vs 5·2 ±0·79 (mean ± s.e.m.) fmol/mg protein). Autoradiographical localization showed that both OT and AVP receptors were present in the interstitial spaces in the testis consistent with binding to Leydig cells. AVP receptors were also localized on the epithelial surfaces of the seminiferous tubules and on testicular blood vessels. This study has, for the first time, found OT receptors in the testis of the rat which have similar ligand-binding characteristics to mammary and uterine OT receptors. The receptor localizations are consistent with binding to Leydig cells. Journal of Endocrinology (1994) 141, 343–352