Accumulated evidence suggests that neurosteroids modulate GABAA receptors through binding interactions with transmembrane domains. To identify these neurosteroid binding sites directly, a neurosteroid-analog photolabeling reagent, (3α,5β)-6-azipregnanolone (6-AziP), was used to photolabel membranes from Sf9 cells expressing high-density, recombinant, His8-β3 homomeric GABAA receptors. 6-AziP inhibited 35S-labeled t- butylbicyclophosphorothionate binding to the His8-β3 homomeric GABAAreceptors in a concentration-dependent manner (IC50 = 9 ± 1 μM), with a pattern consistent with a single class of neurosteroid binding sites. [3H]6-AziP photolabeled proteins of 30, 55, 110, and 150 kDa, in a concentration-dependent manner. The 55-, 110-, and 150-kDa proteins were identified as His8-β3 subunits through immunoblotting and through enrichment on a nickel affinity column. Photolabeling of the β3 subunits was stereoselective, with [3H]6-AziP producing substantially greater labeling than an equal concentration of its diastereomer [3H](3β,5β)-6-AziP. High-resolution mass spectrometric analysis of affinity-purified, 6-AziP-labeled His8-β3 subunits identified a single photolabeled peptide, ALLEYAF-6-AziP, in the third transmembrane domain. The identity of this peptide and the site of incorporation on Phe301 were confirmed through high-resolution tandem mass spectrometry. No other sites of photoincorporation were observed despite 90% sequence coverage of the whole β3 subunit protein, including 84% of the transmembrane domains. This study identifies a novel neurosteroid binding site and demonstrates the feasibility of identifying neurosteroid photolabeling sites by using mass spectrometry.