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 α1β1γ2, α1β3γ2 and α6β3γ2 GABAA receptor subtypes. The chemical agent utilized was hydroxylamine, whose substrate is a relatively rare asparagine-glycine amide bond that occurs only in the α 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 α1 subunit-containing receptors, incorporation of [3H]flunitrazepam occurs within residues 1–103 of the α1 subunit, while incorporation of [3H]Ro15-4513 occurs within the region of the α1 subunit that lies between residue 104 and the C-terminus. Photolabelling of membranes prepared from the α6β3γ2-expressing cell line with [3H]Ro15-4513 resulted in the incorporation of radiolabel into two major protein species of Mr 56 000 and Mr 48 000, indicating incorporation into the α6 subunit and possibly also the γ2 subunit. Hydroxylamine cleavage of α6-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. Copyright © 1996 Elsevier Science Ltd