GH-releasing hormone (GHRH)-like mRNA and immunoreactivity (t-GHRH-li) are present in the testes of rats and humans. To learn more about the physiology of t-GHRH-li mRNA, we performed a series of experiments that disrupted various testicular functions. We employed ethylene dimethanesulfonate, a Leydig cell toxin, to assess the effects of Leydig cell ablation on t-GHRH-li mRNA and protein levels in prepubertal and postpubertal male rats. The ethylene dimethanesulfonate treatment resulted in decreases in serum testosterone, but had no effect on t-GHRH-li mRNA or peptide levels. To assess the effect of GHRH on Leydig cell steroidogenesis, Leydig cells were isolated by Percoll gradient centrifugation and cultured in the presence or absence of hCG, GHRH, or hCG plus GHRH. GHRH had no effect on steroidogenesis by Leydig cells, either alone or in combination with hCG. To localize t-GHRH-li mRNA within rat testis, in situ hybridization analysis was performed on testicular sections from normal rats, using a [35S]GHRH riboprobe. Grains were detected in spermatogenic cells with the antisense probe, whereas none was detected with the sense strand (control) probe. To verify these results, Northern blot analysis of RNA from separated testicular cells was performed, t-GHRH-li mRNA was detected in spermatocytes and round spermatids and to a lesser extent in Sertoli cells, but not in elongating spermatids, Leydig cells, or peritubular myoid cells. t-GHRH-li mRNA was also not found in epididymis. Since our experiments localized t-GHRH-li mRNA to spermatogenic cells, methoxyacetic acid (MAA), a pachytene spermatocyte toxin, was administered to postpubertal rats to determine whether t-GHRH-li is expressed primarily in pachytene spermatocytes. MAA treatment caused a decrease in testicular weight, which gradually returned to control levels by 42 days. Serum FSH levels in the treated animals fluctuated over the course of the experiment, while those in control animals remained steady. However, there was no difference in testicular GHRH-li mRNA levels between control and treated animals at any treatment time. Insulin-like growth factor-I and -II mRNA levels were also unaltered by the MAA treatment. We conclude from these results that t-GHRH-li is synthesized in early spermatogenic cells, but not in mature sperm, and that testicular GHRH-li does not play a major role in steroidogenesis by the Leydig cell.