The GABAA receptor in brain membranes prepared from bovine cerebral cortex and cerebellum has been photoaffinity-labelled by the classical benzodiazepine agonist, [3H]flunitrazepam, or by the partial inverse agonist [3H]Ro 15-4513. Following solubilization and precipitation with trichloroacetic acid, the photoaffinity-labelled receptor preparations were subjected to specific chemical cleavage using hydroxylamine, a reagent which cleaves specifically at a relatively rare Asn-Gly bond. The resulting peptides were resolved by denaturing polyacrylamide gel electrophoresis and mapping of these peptides to the known amino acid sequences of the GABAA receptor subunits has localized the photoaffinity-labelling sites for these two ligands to distinct portions of the alpha subunits. It is shown that the site for [3H]flunitrazepam photoaffinity-labelling in the receptor populations of both the cerebral cortex and cerebellum occurs within residues 1-103 of the bovine alpha 1 subunit sequence (or within analogous segments of homologous alpha subunits). In contrast, the site of photoaffinity-labelling by [3H]Ro 15-4513 in the cerebral cortex and in the diazepam-sensitive GABAA receptor population of the cerebellum lies between residues 104 and the carboxy-terminus of the bovine alpha 1 or homologous alpha subunits. However, the [3H]Ro 15-4513 photoaffinity-labelling site for the diazepam-insensitive receptors of the cerebellum is shown to occur within residues 1-101 (alpha 6 subunit numbering). These results demonstrate that the photoaffinity-labelling sites for [3H]flunitrazepam and [3H]Ro 15-4513 on the GABAA receptor are localized to distinct domains of the alpha 1 subunit and that [3H]Ro 15-4513 photoaffinity labels a site on the alpha 6 subunit that is unique from its site of labelling on the alpha 1 subunit.