The ρ1 GABA receptor is inhibited by a number of neuroactive steroids. A previous study (J Pharmacol Exp Ther 323:236-247, 2007) focusing on the electrophysiological effects of inhibitory steroids on the ρ1 receptor found that steroid inhibitors could be divided into three major groups based on how mutations to residues in the M2 transmembrane domain modified inhibition. It was proposed that the steroids act through distinct mechanisms. We selected representatives of the three groups (pregnanolone, tetrahydrodeoxycorticosterone, pregnanolone sulfate, allopregnanolone sulfate, and ρ-estradiol) and probed how these steroids, as well as the nonsteroidal inhibitor picrotoxinin, modify GABA-elicited fluorescence changes from the Alexa 546 C5 maleimide fluorophore attached to residues in the extracellular region of the receptor. The fluorophore responds with changes in quantum yield to changes in the environment, allowing it to probe for structural changes taking place during channel activation or modulation. The results indicate that the modulators have specific effects on fluorescence changes suggesting that distinct conformational changes accompany inhibition. The findings are consistent with the steroids acting as allosteric inhibitors of the ρ1 GABA receptor and support the hypothesis that divergent mechanisms underlie the action of inhibitory steroids on the ρ1 GABA receptor.