The r1 GABAA receptor is prominently expressed in the retina and is present at lower levels in several brain regions and other tissues. Although the r1 receptor is insensitive to many anesthetic drugs that modulate the heteromeric GABAA receptor, it maintains a rich and multifaceted steroid pharmacology. The receptor is negatively modulated by 5b-reduced steroids, sulfated or carboxylated steroids, and b-estradiol, whereas many 5a-reduced steroids potentiate the receptor. In this study, we analyzed modulation of the human r1 GABAA receptor by several neurosteroids, individually and in combination, in the framework of the coagonist concerted transition model. Experiments involving coapplication of two or more steroids revealed that the receptor contains at least three classes of distinct, nonoverlapping sites for steroids, one each for the inhibitory steroids pregnanolone (3a5bP), 3a5bP sulfate, and b-estradiol. The site for 3a5bP can accommodate the potentiating steroid 5aTHDOC. The findings are discussed with respect to receptor modulation by combinations of endogenous neurosteroids.