Estrogenic metabolites of circulating androgens have important effects on the organization and activation of neural circuits controlling reproductive behavior and physiology in males of many vertebrate species. Previous studies indicate that aromatase, the enzyme that converts androgens to estrogens, is expressed most abundantly in neurons in limbic brain regions. Songbirds are unique in that aromatase is expressed at unusually high levels throughout the telencephalon of both males and females. We assume that estrogens formed in the telencephalon itself masculinize neural circuits controlling song, since the brain is a major source of circulating estrogens in adult males. However, the cellular localization of telencephalic aromatase in songbirds remains unknown. We have established primary cultures from telencephalons of developing zebra finches and found aromatase activity (conversion of 3H-androstenedione or 3H-testosterone to 3H-estrone plus 3H-estradiol) at some of the highest levels reported for brain tissue of any species. Both neurons and glia were identified in these cultures based on cell morphology and labeling by specific immunohistochemical markers. However, when culture conditions were manipulated to reduce the incidence of either neurons or glia by varying the age of cultures or their plating density, treating with the neurotoxin kainic acid, physically shaking off loosely attached neurons, or preparing cultures in media that encouraged enrichment of neurons, high levels of aromatase persisted. Furthermore, Northern blot analysis of total RNA extracted from enriched neuronal or glial cultures indicated the presence of aromatase mRNA in both cell preparations. In situ hybridization with a zebra finch aromatase cDNA probe conjugated to digoxigenin showed the cultures contained darkly labeled neurons and lightly labeled non-neuronal cells, presumably astrocytes. We conclude that aromatase is expressed in both neuronal and non-neuronal cells in these cultures, suggesting that both cell types may also express the enzyme in vivo. The presence of aromatase outside of neurons suggests that glia may be targets of estrogen action or that glia may supply some estrogen to the estrogen-sensitive neural circuits in this species.