Chlordiazepoxide (CDPX) enhanced the rate of chloride exchange mediated by the major GABAA receptor found on sealed native membrane vesicles from rat cerebral cortex. The initial rate constant for chloride exchange for this receptor, (JA), a measure of open channel, was determined from the progress of GABA-mediated influx of 36Cl-. The dependence of JA on GABA concentration was hyperbolic in the presence of CDPX (150 microM, sufficient to give maximum enhancement of chloride exchange rate) but sigmoid in its absence. Enhancement of channel opening (10-fold at 0.3 microM GABA) decreased with increasing GABA concentration. The maximal response, above 1,000 microM GABA, was unaltered. The half-response concentration was reduced from 80 microM to 50 microM. CDPX alone caused no measurable 36Cl- exchange. In the presence of CDPX, channel opening occurred with only one bound GABA molecule, whereas in its absence, channel opening with two bound GABA molecules was much more favorable. This could not be direct allosteric modulation of the channel opening conformational change by binding of CDPX at effector sites, but could be explained by an additional change of the receptor on binding CDPX to give a closed state which gave channel opening mediated by a single GABA binding site. Another possibility is that CDPX could act at one of the channel opening binding sites without a postulated, second closed conformational state.