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The neurohypophysial peptide hormone oxytocin is associated with parturition/milk ejection. The identification of a non-neural source of oxytocin secreted in significant amounts from the ovarian corpus luteum of ruminants stimulated interest in this octapeptide hormone. Oxytocin has an ovarian source, whereas an oxytocin-like peptide is reportedly present in the testes. Highly purified populations of guinea pig Leydig cells were incubated with a maximally stimulating dose of 100 ng/ml LH for 24 hours in the presence of increasing concentrations of sodium ascorbate. Sample supernatants were extracted, concentrated under vacuum and reconstituted with acidified absolute ethanol. Samples were analyzed for oxytocin using high performance liquid chromatography HPLC with electrochemical detection and known concentrations of an authentic oxytocin standard. Leydig cells stimulated with 0, 25 and 50 uM ascorbate produced and secreted 40 +/- 1.2, 77 +/- 14 and 74 +/- 26 pg of an oxytocin-like peptide, respectively, per 1 x 106 cells. It would appear that guinea pig Leydig cells are capable of producing an oxytocin-like peptide de novo. Low concentrations of ascorbate stimulate the production of this peptide in Leydig cells cultured in vitro. Comparison of the amino acid composition and sequence of this oxytocin-like peptide with authentic oxytocin are already in progress to confirm whether Leydig cells possess the biosynthetic machinery for testicular oxytocin production.