Aging-related alterations in both protein and mRNA expression of gamma 2S and gamma 2L subunits of the GABAA receptors have been observed in several brain areas of Sprague-Dawley and Fischer 344 rats. Subunit-specific antibodies to gamma 2S and gamma 2L as well as a riboprobe to the large intracellular loop of gamma 2, which recognizes both gamma 2S and gamma 2L mRNAs, in conjunction with computerized image analysis were used for quantitative immunocytochemistry and in situ hybridization. In addition, specific oligonucleotide probes to gamma 2S or gamma 2L mRNA were used for quantitative dot blot hybridization. A large increase in the number of heavily immunostained neurons with the anti-gamma 2L antibody was detected in the cerebral cortex (115%) of old rats. However, only a small (but significant) aging-related increase in the density of gamma 2L immunostaining (7%) was observed throughout the cerebral cortex whereas no significant aging-related change in gamma 2L mRNA was detected in this brain region. Contrary to gamma 2L, the gamma 2S immunostaining did not show aging-related increased number of heavily immunostained neurons in cerebral cortex. Moreover, the density of gamma 2S immunostaining and the expression of gamma 2S mRNA were significantly decreased in the cerebral cortex (9-24%). Important aging-related changes were also found in the cerebellum of old rats where the expression of both gamma 2S and gamma 2L peptides was significantly decreased (24% and 23% respectively). This decrease in gamma 2 protein expression was accompanied by decreased expression of gamma 2S (16-38%) and gamma 2L (24%) mRNAs. Nevertheless, the most important decrease of gamma 2S (48%) and gamma 2L protein (20%) was revealed in the molecular layer of the cerebellum. In addition, the expression of gamma 2S protein was increased (14%) whereas the expression of gamma 2L was decreased (13%) in the granule cell layer. Therefore, the relative expression of gamma 2S protein in both layers was reversed in old animals. The observed aging-related changes in the expression of GABAA receptor subunits might lead to altered GABAA receptor/benzodiazepine receptor subunit composition.