Subunit-specific antibodies to all the gamma subunit isoforms described in mammalian brain (gamma(1), gamma(2S), gamma(2L), and gamma(3) have been made. The proportion of GABA(A) receptors containing each gamma subunit isoform in various brain regions has been determined by quantitative immunoprecipitation. In all tested regions of the rat brain, the gamma(1) and gamma(3) subunits are present in considerable smaller proportion of GABA(A) receptor than the gamma(2) subunit. Immunocytochemistry shows that gamma(1) immunoreactivity concentrates in the stratum oriens and stratum radiatum of the CA1 region of the hippocampus. In the dentate gyrus, gamma(1) immunoreactivity concentrates on the outer 2/3 of the molecular layer coinciding with the localization of the axospinous synapses of the perforant pathway. In contrast, gamma(3) immunoreactivity concentrates on the basket cells and other GABAergic local circuit neurons of the hilus. These cells are also rich in gamma(2S). In the cerebellum, gamma(1)++ immunolabeling was localized on the Bergmann glia. The gamma(2S) and gamma(2L) subunits are differentially expressed in various brain regions. Thus the gamma(2S) is highly expressed in the olfactory bulb and hippocampus whereas the gamma(2L) is very abundant in inferior colliculus and cerebellum, particularly in Purkinje cells, as immunocytochemistry, in situ hybridization and immunoprecipitation techniques have revealed. The gamma(2S) and gamma(2L) coexist in some brain areas and cell types. Moreover, the gamma(2S) and gamma(2L) subunits can coexist in the same GABA(A) receptor pentamer. We have shown that this is the case in some GABA(A) receptors expressed in cerebellar granule cells. These GABA(A) receptors also have alpha and beta subunits forming the pentamer. Immunoblots have shown that the rat gamma(1), gamma(2S), gamma(2L) and gamma(3) subunits are peptides of 47, 45, 47 and 44 kDa respectively. Results also indicate that there are aging-related changes in the expression of the gamma(2S) and gamma(2L) subunits in various brain regions which suggest the existence of aging-related changes in the subunit composition of the GABA(A) receptors which in turn might lead to changes in receptor pharmacology. The results obtained with the various gamma subunit isoforms are discussed in terms of the high molecular and binding heterogeneity of the native GABA(A) receptors in brain.