In an attempt to determine the time of origin and the final localization of Purkinje cells in the cerebellum, pregnant mice were injected with tritiated thymidine successively, four to six times daily, beginning on either day 10, 11, 12, 12.5, 13, or 14 of gestation, considering 6 a.m. on the day when vaginal plugs were found as day 0. Offspring were killed at 30 days of age, and serial sagittal sections of the cerebellum were prepared for autoradiography. Labeled and unlabeled Purkinje cells in the various regions of the cerebellum were counted, and unlabeled ones were considered to be formed earlier than the initiation of the injections. In the medial level of the vermis, the majority of the Purkinje cell population was formed on day 12 of gestation and a few on day 13, whereas the cells in the paravermian and hemispheric portions were formed on days 11 and 12. Thus, a slight lateromedial gradient of the time of Purkinje cell origin was demonstrated. Purkinje cells that were formed after day 12.5 were distributed more in the vermis than in the hemisphere, more in the ventral region of the vermis (lingula, ventral lobule of lobulus centralis, and nodulus) than in the dorsal region (culmen, declive, tuber, pyramis, and uvula), and deep in the vermian fissures, rather than on the surface of the lobules. These findings suggest that postnatally, early maturing regions of the cerebellum may have received many of the later formed Purkinje cells.