The interaction of three types of steroids with the GABAA recognition site labeled by the antagonist ligand [3H]SR 95531 was evaluated in rat brain cortical membranes. The first type is the GABA site antagonist RU 5135, which potently (IC50 7 nM) but also incompletely (Imax 82%) displaced [3H]SR 95531. RU 5135 probably binds only to high affinity [3H]SR 9553] sites recognized by GABA and unlabelled SR 95531. The second type are the neuroactive steroids which act as positive allosteric modulators, including 3 alpha-hydroxy-5 beta-pregnan-20-one (3 alpha, 5 beta-P) and 5 beta-tetrahydrodeoxycorticosterone (5 beta-THDOC), which inhibited [3H]SR 95531 binding with limited efficacy (IC50 460 nM and 1.4 microM, Imax 41 and 31%, respectively). In contrast, 3 alpha-hydroxy-5 alpha-pregnan-20-one (3 alpha, 5 alpha-P) was inactive. The third type are the neurosteroids acting as negative allosteric modulators, such as pregnenolone sulfate, which inhibited [3H]SR 95531 binding with limited efficacy (IC50 10 microM, Imax 23%). In the presence of a saturating concentration of pregnenolone sulfate, 3 alpha, 5 beta-P further inhibited [3H]SR 95531 binding suggesting that these two steroids act through different sites or, possibly, at different populations of GABAA receptors. The allosteric modulation was selective for steroids since benzodiazepines and barbiturates were inactive up to 100 microM. Taken together, these data suggest that 3 alpha, 5 beta-P and 5 beta-THDOC modulate [3H]SR 95531 binding by interacting with a unique site on the GABAA receptor complex distinct from the sites for 3 alpha, 5 alpha-P, pregnenolone sulfate, GABA, benzodiazepines, and barbiturates.