Electrophoretic and morphologic methods have been used to study the time course of [35S] methionine-labeled proteins accumulated in the incubation medium of rat fetal testes and seminiferous cords/tubules during their development. We have found that Sertoli cell-specific secretory proteins S70, S45 and S35 became progressively prominent as premeiotic, meiotic and postmeiotic spermatogenic events were established in the seminiferous tubules. In the sexually mature rat, S70, S45 and S35 were expressed in a spermatogenic stage-dependent manner. While S70, S45 and S35 were present in Stage VII-VIII, S45 and S35 were observed in Stages X and XIV. Neither S70, S45 nor S35 were detected in Stage IV. A relevant group of high molecular weight proteins, previously reported as characteristic products of cultured peritubular cells, accumulated in the incubation medium of seminiferous cords from postnatal Day 0-15 rats. This group of high molecular weight proteins appears when peritubular cells are proliferative and are engaged in the organization of the seminiferous tubular wall. A low molecular weight protein, designated T35, was also detected. T35 was prominent in the medium of incubated fetal testes and seminiferous cords of postnatal rats 0- to 5-days-old and disappeared gradually thereafter. A set of proteins (designated SP1 and SP2) previously ascribed to both cultured Sertoli and peritubular cells, were recognized during the early postnatal stages of seminiferous tubular development. SP1 and SP2 displayed age-dependent fluctuations in their [35S] methionine labeling. The timing of appearance of S70, S45, and S35 indicates both age- and spermatogenic stage-related activity that, in the future, may prove to be functionally significant in the spermatogenic process.