We have studied the posttranslational processing of prohuman GH-releasing hormone (pro-hGRH) to the mature hormones, hGRH(l-44)-NH2and hGRH(l-40)-OH, and its carboxyl-terminal peptide (hGCTP) in pituitary cells from transgenic mice bearing a metallothionein-hGRH fusion gene after incubation with [35S]methionine. After separation on HPLC,35S-labeled and unlabeled hGRH in medium and cell extracts were characterized by RIA and immunoprecipitation with antisera against hGRH and against hGCTP. After a 4-h pulse, unlabeled and [35S]pro-hGRH, hGRH(l-44)-NH2, and hGRH(l-40)-OH were identified in medium and cell extracts by both RIA and immunoprecipitation with anti-hGRH serum. In cell extracts, after a 0.5-h pulse, [35S]pro-hGRH and hGRH(l-44)-NH2but not [35S]hGRH(l-40)-OH were detectable. After a 0.5-h chase, however, 35S-labeled hGRH(l-40)-OH, pro-hGRH, and [35S]hGRH(l-44)-NH2were all measurable. After a 4-h chase, comparable levels of [35S]hGRH(l-44)-NH2and hGRH(l-40)-OH were present, and very little intracellular35S-pro-hGRH remained. A progressive decrease in the ratio of immunoprecipitable pro-hGRH to mature hGRH peptides and an increase in the ratio of hGRH(l-40)-OH to hGRH(l-44)-NH2was observed in the two chase periods. In medium, [35S] hGRH(l-44)-NH2was detectable at all times, whereas only minimal amounts of [35S]hGRH(l-40)-OH were present. Labeled and unlabeled pro-hGRH in cell extracts was also detected with anti-hGCTP serum, and another peak, which coeluted with synthetic hGCTP, was also identified. The low molar ratio of intracellular immunoreactive hGCTP to hGRH (<0.02) suggests a more rapid turnover rate of hGCTP than of hGRH. These results demonstrate the processing of hGRH prohormone to both mature forms of hGRH and provide evidence that hGRH(1-40)-OH is derived from hGRH(l-44)-NH2.