Activin and inhibin are peptide hormones produced in the gonads which may act as autocrine and/or paracrine regulators of testicular function. Sertoli cells produce inhibin, and it has recently been shown that Leydig cells can produce activin in vitro. To further explore the local actions of activin and inhibin in the testis, Sertoli and germ cells were isolated from immature rats and cocultured in vitro. In these cultures we demonstrate that activin A and activin B, but not inhibin A, stimulated spermatogonial proliferation in vitro. Activin increased [3H]thymidine incorporation 2- to 4-fold in cocultures after 48-72 h of treatment. Using autoradiography, the label was localized in the clusters of spermatogonia adhering to the Sertoli cell monolayer. Additionally, activin stimulated a reaggregation of the cultures into tubule-like structures. Fluorescence-activated cytometry was used to analyze the cell population based on size, DNA content, and lipid content. Sertoli cells were identified using Nile Red staining of intracellular lipid droplets; spermatogonia are Nile Red-negative. Activin treatment caused a marked increase in the fraction of Nile Red-negative cells in the cocultures. Activin also caused an increase in the percentage of these cells having 4C DNA. Lastly, specific binding of activin A to 2C, but not 4C, germ cells was demonstrated. These data demonstrate that activin acts as a regulator of spermatogonial proliferation in the male.