GABA-mediated chloride influx into rat cerebral cortical microsacs, and its potentiation by pentobarbital (PB), was studied in incubations lasting 50 or 3000 ms. The 3000 ms assays were carried out manually, while the 50 ms assays were performed in a quench flow machine. The EC50 of the GABA effect and the Hill coefficient were both lower at the longer incubation times. Percent increase of the effect of GABA (at EC30 concentration) by pentobarbital was greater in the 3000 ms than in the 50 ms incubations. Preincubation of microsacs with PB for 5 s before the 50 ms incubation with GABA, did not increase the effect of PB beyond that obtained by adding PB into the incubation only. This finding is consistent with earlier ones cited, which imply that only PB binding that occurs after GABA has bound can potentiate the effect of GABA, and suggests that the concentrations of PB and GABA required to enhance chloride influx in vivo may be higher than those commonly reported in the literature.