Recent studies have shown that both proopiomelanocortin (POMC)-derived peptides and a range of POMC gene transcripts are present in the testis. Previous immunocytochemical studies have reported immunoreactive (ir)-beta-endorphin (EP) and ir-ACTH to be localized in the Leydig cells, and ir-NacEP in spermatogonia and primary spermatocytes. In the present study, we have further examined the hypothesis that testicular Leydig cells are the principal site of synthesis of these peptides, by determining the effects of the administration of the cytotoxic drug ethane dimethane sulphonate (EDS) which selectively destroys the Leydig cells of the testis. As expected, serum testosterone levels fell and serum FSH/LH levels increased within 3 days of EDS administration, returning to normal levels 4-8 weeks later. In contrast, the testicular content of POMC-derived peptides and POMC mRNA levels in these animals was not significantly altered throughout the experimental period. In addition, POMC mRNA was not detected in a purified Leydig cell preparation derived from adult male rats, and POMC-derived peptides were also undetectable in the media of a similar preparation following cell culture. These data suggest that in the adult the predominant site of rat POMC gene expression is in testicular interstitial cells other than Leydig cells.