Classical benzodiazepines, which are widely used as sedatives, anxiolytics and anticonvulsants, exert their therapeutic effects through interactions with heteropentameric GABA(A) receptors composed of two alpha, two beta and one gamma 2 subunit. Their high affinity binding site is located at the interface between the gamma 2 and the adjacent alpha subunit. The alpha-subunit.gene family consists of six members and receptors can be homomeric or mixed with respect to the alpha-subunits. Previous work has suggested that benzodiazepine binding site ligands with selectivity for individual GABA(A) receptor subtypes, as defined by the benzodiazepine-binding alpha subunit, may have fewer side effects and may even be effective in diseases, such as schizophrenia, autism or chronic pain, that do not respond well to classical benzodiazepines. The distributions of the individual alpha subunits across the CNS have been extensively characterized. However, as GABA(A) receptors may contain two different alpha subunits, the distribution of the subunits does not necessarily reflect the distribution of receptor subtypes with respect to benzodiazepine pharmacology. In the present study, we have used in vivo [F-18]flumazenil PET and in vitro [H-3] flumazenil autoradiography in combination with GABA(A) receptor point-mutated mice to characterize the distribution of the two most prevalent GABA(A) receptor subtypes (alpha 1 and alpha 2) throughout the mouse brain. The results were in agreement with published in vitro data. High levels of alpha 2-containing receptors were found in brain regions of the neuronal network of anxiety. The alpha 1/alpha 2 subunit combinations were predictable from the individual subunit levels. In additional experiments, we explored in vivo [F-18]flumazenil PET to determine the degree of receptor occupancy at GABA(A) receptor subtypes following oral administration of diazepam. The dose to occupy 50% of sensitive receptors, independent of the receptor subtype(s), was 1-2 mg/kg, in agreement with published data from ex vivo studies with wild type mice. In conclusion, we have resolved the quantitative distribution of alpha 1- and alpha 2-containing homomeric and mixed GABA(A) receptors in vivo at the millimeter scale and demonstrate that the regional drug receptor occupancy in vivo at these GABA(A) receptor subtypes can be determined by [F-18]flumazenil PET. Such information should be valuable for drug development programs aiming for subtype-selective benzodiazepine site ligands for new therapeutic indications.