Immunohistochemical evidence has indicated that beta-endorphin (beta EP) is present in the Leydig cells of fetal, neonatal, and adult mice and hamsters. In vivo experiments suggest that hCG and/or testosterone may increase the synthesis and release of the peptide from the Leydig cell compartment. Since cultured fetal Leydig cells have considerable potential for long term studies and elucidation of trophic hormone actions in vitro, we evaluated beta EP production in this system. Fetal Leydig cells were maintained in culture for 5 days in medium with 1 microgram ovine LH added every third day in the presence or absence of inhibitors of cholesterol (aminoglutethimide) or pregnenolone metabolism (cyanoketone and spironolactone), or known regulators of beta EP production [dexamethasone (DEX)]. Media were assayed for testosterone and beta EP by RIA methods. Beta EP accumulation over 3 and 5 days was markedly increased by inhibitors of steroid biosynthesis (1.5-fold) and reduced by DEX only after treatment for 5 days (by 50%). Acute hCG stimulation significantly increased beta EP levels by 5- to 9-fold in all conditions tested. Inhibition of Leydig cell steroid biosynthesis markedly increased basal and hCG-stimulated beta EP output (by 150-200%). In contrast, DEX reduced basal and hCG-stimulated beta EP production (by approximately 50%). HPLC analysis of cultured pooled media revealed that the beta EP immunoreactivity eluted at the retention time of authentic rat beta EP. The pattern of beta EP stimulation was not reflected by testosterone levels that were low or undetectable in controls and under conditions in which spironolactone/cyanoketone or aminoglutethimide were present; most importantly, inhibition of steroid biosynthesis markedly increased beta EP levels. In addition, beta EP (10(-7) M) did not affect testosterone production, and opiate binding was not detected on Leydig cells. The lack of degradation of this opioid peptide in the fetal cultures contrasted with results from adult cultures and provided an ideal system for studies of the regulation of this peptide in Leydig cells. These results demonstrate that beta EP is released from fetal Leydig cells in culture and that acute stimulation of Leydig cells by hCG can enhance beta EP secretion. These changes are not mediated by testosterone. In contrast, testosterone or its metabolites may exert negative autocrine modulation of beta EP production.(ABSTRACT TRUNCATED AT 400 WORDS)