We have used [3H]flunitrazepam and [3H]Ro15-4513 as photoaffinity labelling agents in combination with a chemical cleavage technique to localize the benzodiazepine recognition sites of specific human recombinant alpha 1 beta 1 gamma 2, alpha 1 beta 3 gamma 2 and alpha 6 beta 3 gamma 2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the alpha subunits of the receptors examined in this study. Cleavage products were resolved using sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). The results of these experiments show that, in the alpha 1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1-103 of the alpha 1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the alpha 1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the alpha 6 beta 3 gamma 2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of M(r) 56,000 and M(r) 48,000, indicating incorporation into the alpha 6 subunit and possibly also the gamma 2 subunit. Hydroxylamine cleavage of alpha 6-containing receptors labelled with [3H]Ro15-4513 produced a gel profile consistent with the incorporation of the label occurring between residue 125 and the C-terminal. Thus, we have shown that the recognition sites for the agonist [3H]flunitrazepam and the inverse agonist [3H]Ro15-4513 occur within distinct domains of the human GABAA receptor.