Endogenous opioid systems (i.e. opioids and opioid receptors) play a role in regulating neural development. Using the cerebellar cortex of 6-day-old rats, the most potent opioid peptides involved with cell proliferation were assessed. In both the external germinal (granule) layer (EGL), a germinative matrix giving rise to neurons, and the medullary layer (MED), a pool of cells that are the precursors of glia (astrocytes and oligodendrocytes), [Met5]enkephalin and peptide F were extremely potent in depressing the labeling index (LI) using [3H]thymidine and autoradiographic techniques; concentrations as low as 100 micrograms/kg reduced the LI of EGL cells by 24% and MED cells by 43%. This inhibition of DNA synthesis by opioid peptides was blocked by concomitant exposure to to naloxone, an opioid antagonist. Peptide action was apparent 2 h following drug administration, and concentrations of 80 micrograms/kg but not 1 or 10 micrograms/kg [Met5]enkephalin depressed the LI. These results identify a selective group of opioid peptides, derived from proenkephalin A, as the potent, natural, inhibitory factors targeted to cell proliferation of cells destined to be neurons and glia in the developing nervous system.