Newborn rat cerebellum microexplants cultured in Minimal Essential Medium with glucose and insulin released plasminogen activator (PA), which was detected in living cultures by a substrate overlay assay. Gel electrophoresis of cerebellum-conditioned medium followed by zymography resolved PA activity in two separate bands of 48,000 and 75,000 daltons apparent mol. wt. Using specific antisera, these bands were shown to be respectively urokinase and tissue-type PA. Cerebellum conditioned medium as well as purified human urokinase induced the proliferation and outgrowth of glial fibrillary acid protein-positive cells from newborn cerebellar microexplants. The effect was suppressed by the serine protease inhibitor phenyl methanesulfonylfluoride. Since PAs are most likely of neuronal origin, we suggest that at least one of these proteases acts as a neuronoglial mitogenic signal during development.