Evidence suggests that systemic oxytocin (OT) causes contractions of the prostate gland during ejaculation in eutherians, although functional OT receptors in this tissue have not been identified. Male marsupials secrete mesotocin (MT) from the pituitary and have relatively large, muscular prostate glands, so we examined MT receptors (MTRs) in the reproductive tract of the male tammar wallaby at the mRNA and protein level. We first obtained a partial (588 base pair) sequence of the tammar MTR cDNA that showed high homology to eutherian OT receptors (74-77%) and low homology to vasopressin receptors (38-52%). Analysis by reverse transcription-polymerase chain reaction demonstrated MTR mRNA in the adult, juvenile, and pouch young prostate and epididymis, but not testis. MTR transcripts were observed in the smooth muscle layers surrounding the urethral lumen and in the fibromuscular capsule. There was a single high-affinity 125I-D(CH2)5[Tyr(Me)2, Tyr4, Orn8, Tyr-NH29]-vasotocin (125I-OTA) binding site in the adult prostate. Competitive binding assays revealed identical ligand-binding profiles to the myometrium MTR (OTA > OT = MT > arginine vasopressin [AVP] antagonist > AVP). A lower-affinity 125I-OTA-binding site was present in the testis, with ligand-binding profiles indicating binding to vasopressin receptors. MTR concentrations in the prostate were 8-fold lower than concentrations in the myometrium. Our data demonstrate the presence of an MTR gene and functional receptor protein in the prostate gland, but not the testis, of the tammar. Localization of MTRs to the smooth muscle fibers in the capsule and surrounding the urethral lumen suggests a contractile function for MT during ejaculation.