Bulk-isolated microglial cells from the adult rat brain grown in N2 medium supplemented with 10% fetal calf serum survived for at least 2 weeks, and their purity was >99% at day 1 and >93% at day 7. The phenotype of freshly plated cells was comparable to that of "resting," ramified microglia in vivo. With time in culture and with different schedules, depending on the parameter considered, microglia acquired antigenic (e.g., positivity for vimentin, ED1, major histocompatibility complex class I antigens, leukocyte common antigen, and to a lesser extent CD4) and functional (e.g., proliferation, phagocytosis) features characteristic of "activated" microglia as described in situ. Production of nitrite and prostaglandin E2 in response to lipopolysaccharide increased greatly with time in culture. Phagocytosis was also accompanied by increased release of nitrite and prostaglandin E2, the latter being more affected than the first by the age of the cultures. The culture system described may be suitable to study the factors that can modulate "activation" of adult microglia.