A procedure is described for the preparation and maintenance of human Sertoli-spermatogenic cell cocultures using biopsies of normal and undescended testis. The evaluation of cell viability and differentiation potential of cultured spermatogenic cell was monitored by [3H]thymidine labeling combined with light microscopic autoradiography. Spermatogenic cells of the same progeny, connected by intercellular bridges, display synchronous DNA synthesis when labeled at the preleptotene stage of meiotic prophase. The pattern of [35S]methionine-labeled secretory proteins was determined by two-dimensional electrophoresis and autoradiography during testicular development and compared with these observed in human Sertoli-spermatogenic cell cocultures prepared from same specimens. Both testicular tissue and cocultured Sertoli and spermatogenic cells displayed comparable patterns of secretory proteins. A discrete group of acidic polypeptides of Sertoli cell origin enhanced their radiolabeling intensity during testicular development. Results of this paper indicate that human Sertoli-spermatogenic cell cocultures could be valuable for assessing the proliferation and differentiation potential of spermatogenic cells in children with cryptorchid testis.