Recent studies indicate that proopiomelanocortin (POMC)-derived peptides are present in the testis and may be involved in the regulation of gonadal function, although their precise role still remains obscure. In the present study, we investigated in the rat whether testicular interstitial fluid (TIF), a functionally important compartment of the testis, contains POMC-derived peptides. Adult rats were used for all the experiments, and TIF was collected from each individual testis and analyzed for the presence of ACTH and beta-endorphin-like immunoreactivity (beta end-LI). Initial studies indicated that both ACTH and beta end-LI can be readily detected in TIF from intact rats, and that the concentrations of these peptides are severalfold higher than those in the peripheral plasma of the same animals. Similar studies in rats 3-5 days after hypophysectomy indicate that although ACTH and beta end-LI levels in plasma were undetectable, high levels of both peptides were measured in TIF. Moreover, serial dilution curves of TIF showed parallelism with the respective standard curves in the beta end and ACTH assays. Additional studies indicated that levels of POMC peptides in TIF were modified by treatments that affect the endocrine function of the testis. In that respect, hCG given sc to hypophysectomized rats increased testosterone levels in TIF and plasma, and similarly increased beta end-LI concentrations in TIF. A LHRH analog (LHRH-A; [D-Ala6,des-Gly10]LHRH-ethylamide) given sc to hypophysectomized rats resulted in increased testosterone production, but decreased beta end-LI in TIF, suggesting that the effects of hCG and LHRH-A on testicular beta end-LI secretion are not directly coupled with testosterone production. Prolonged treatment of intact rats with the LHRH-A for 2-10 days resulted in progressive declines in testicular weight and testosterone levels in TIF and in plasma, and a significant suppression of beta end-LI levels in TIF as early as 2 days after analog treatment. On the other hand, acute intratesticular injection of LHRH-A to intact rats (5 ng into the right testis) produced a short-lived increase in beta end-LI in TIF 2 h after injection, coincident with a temporary decrease in TIF volume. These results indicate that the POMC-derived peptides ACTH and beta end are present in TIF and are secreted locally into this compartment, supporting previous reports demonstrating the presence and local synthesis of POMC peptides in testicular tissue.(ABSTRACT TRUNCATED AT 400 WORDS)