H-Pro-Leu-Gly-NH2 (PLG) was labeled with 3H-leucine by catalytic tritiation of H-Pro-methylallylglycyl-Gly-NH, and purified by ion-exchange chromatography. Metabolism of 3H-PLG in rat and human plasma was investigated by reversed phase-paired ion high-pressure liquid chromatography. All possible metabolites could be completely separated within 25 min. Half-lives, based on disappearance of intact 3H-PLG, for in vitro metabolism were 26.4 min (rat) and 5.6 days (human). The only significant metabolite was 3H-leucine. A rate-limiting, species-specific enzyme seems responsible for the initial breakdown of PLG. Disappearance of 3H-PLG from rat plasma, following i.v. administration, proceeded with half-lives of 1.03 min (distribution) and 9.8 min (elimination).