To study various aspects of GABAergic metabolism in an easily accessible system, dissociated cells from postnatal rat cerebral cortex were cultured in a serum-based medium and characterized morphologically and biochemically. The majority (70-96%) of the neurons were GABAergic as determined by three double-labeling procedures. The specific activity of glutamine synthetase in the cultures was 4-5% of the levels in rat astrocyte cultures and intact rat brain, indicating that glia were a minor component. The developmental increase of GABA levels preceded the increase of GAD activity in both immunocytochemical and biochemical experiments. GABA turnover rates also increased with culture age and were 20-30% of GAD activity. Four anti-GAD antibodies, which recognize GAD subunits with differing molecular masses to varying degrees, were used to stain cultured neurons and make immunoblots. Immunoblots showed that the neurons contained two major subunits of GAD which differed in mass by 2 kDa. All four antibodies immunostained both neuronal perikarya and neurites but one antibody, which on the immunoblots predominantly labeled the GAD protein with the lower molecular weight, showed a somewhat more pronounced punctate staining, possibly indicating a principal localization to neurites.