Expression of preproenkephalin mRNA by developing glia and neurons was examined in cultures of embryonic and neonatal rat brain. Cultured glia from specific regions of embryonic day 17 and neonatal day 1 rat brain were identified as astrocytes on the basis of both morphology and expression of immunoreactivity for glial fibrillary acidic protein. The level of preproenkephalin mRNA in cultured neonatal hypothalamic astrocytes was comparable to levels present in cultured embryonic striatal and hypothalamic neurons. Levels of the mRNA were significantly higher in astrocytes derived from neonatal hypothalamus compared to astrocytes derived from other areas of the brain. Thus, there is heterogeneity among astrocytes with respect to preproenkephalin expression. Levels of preproenkephalin mRNA in cultured neonatal striatal astrocytes were only one-third as high as levels in embryonic striatal astrocytes; this observation suggests that glial expression of the gene may be down-regulated during development. Although cultured hypothalamic neurons contained substantial levels of prodynorphin mRNA, levels of this mRNA were not detectable in cultured astrocytes from any brain region or in cultured striatal neurons. Thus, glia do not express all opioid peptide genes during development. These observations suggest that expression of the preproenkephalin gene by astrocytes may play a role in development of the brain.