Neuroactive steroids modulate the function of γ-aminobutyric acid, type A (GABAA) receptors in the central nervous system by an unknown mechanism. In this study we have used a novel neuroactive steroid analogue, 3α,5β-6-azi-3-hydroxypregnan-20-one (6-AziP), as a photoaffinity labeling reagent to identify neuroactive steroid binding sites in rat brain. 6-AziP is an effective modulator of GABAA receptors as evidenced by its ability to inhibit binding of [35S]t-butylbicyclophosphorothionate to rat brain membranes and to potentiate GABA-elicited currents in Xenopus oocytes and human endothelial kidney 293 cells expressing GABAA receptor subunits (α1β2γ2). [3H]6-AziP produced time- and concentration-dependent photolabeling of protein bands of ∼35 and 60 kDa in rat brain membranes. The 35-kDa band was half-maximally labeled at a [3H]6-AziP concentration of 1.9 μm, whereas the 60-kDa band was labeled at higher concentrations. The photolabeled 35-kDa protein was isolated from rat brain by two-dimensional PAGE and identified as voltage-dependent anion channel-1 (VDAC-1) by both matrix-assisted laser desorption ionization time-of-flight and ESI-tandem mass spectrometry. Monoclonal antibody directed against the N terminus of VDAC-1 immunoprecipitated labeled 35-kDa protein from a lysate of rat brain membranes, confirming that VDAC-1 is the species labeled by [3H]6-AziP. The β2 and β3 subunits of the GABAA receptor were co-immunoprecipitated by the VDAC-1 antibody suggesting a physical association between VDAC-1 and GABAA receptors in rat brain membranes. These data suggest that neuroactive steroid effects on the GABAA receptor may be mediated by binding to an accessory protein, VDAC-1.