Proliferation of Sertoli cells during fetal and postnatal development of the rat was examined and quantified with light microscope autoradiography. Fetuses in utero were injected subcutaneously with 3H-thymidine. The percentages of Sertoli nuclei that had incorporated label were determined in autoradiographs from fetuses aged 16 through 21 days of gestation. To compare the degree of Sertoli cell proliferation during fetal development with that occurring after birth, pups were also studied at intervals between the day of birth and 3 weeks of age. For each fetus or pup, at least 500 Sertoli cell nuclei in each of three sections were scored as labeled or unlabeled. These data were subjected to analysis of variance and the Newman-Keuls test. The percentage of Sertoli cells incorporating 3H-thymidine increased progressively from day 16 of gestation onward, to a maximum of 26.8% on day 20, two days before birth. Thereafter, this percentage dropped steadily until, in pups 21 days after birth, no labeled Sertoli cells were detected. These findings highlight the fetal period as the time of greatest expansion of the Sertoli cell population and indicate that, at birth, proliferation of these cells is already on the decline.