The biosynthesis of the human DNA repair enzyme uracil-DNA glycosylase has been characterized by the reaction of in vitro- and in vivo-produced protein with an anti-human placental uracil-DNA glycosylase monoclonal antibody. In vitro synthesis of the DNA repair enzyme was examined after the translation of human placental polyadenylated [poly(A)+] RNA by immunoprecipitation of the [35S]methionine-labeled translation products. As defined by sucrose density analysis, immunoprecipitable in vitro products were translated from 16S poly(A)+ RNA and 11S poly(A)+ RNA. While the products of the 11S poly(A)+ RNA were smaller than purified uracil-DNA glycosylase, the product of the 16 S poly(A)+ RNA had a molecular weight of 37,000, identical to the size previously observed for purified human placental uracil-DNA glycosylase. Immunoblot analysis of human placental cell extracts and of normal human fibroblast cell extracts demonstrated the recognition of one Mr 37,000 protein. Immunoprecipitation of [35S]methionine-labeled normal human cell extracts with the anti-glycosylase monoclonal antibody specifically detected only the Mr 37,000 uracil-DNA glycosylase protein. Pulse-chase analysis demonstrated that the 35S radioactivity in the Mr 37,000 uracil-DNA glycosylase decreased over a 5-h interval. These results show that immunoreactive human uracil-DNA glycosylase protein was synthesized at its enzymatically active molecular weight of 37,000 as the primary translation product of a 16S polyadenylated messenger RNA.