The present study was designed to compare the allosteric coupling between the Cl − channel of the GABA A receptor and the different benzodiazepine recognition site subtypes (BZ sites) in the cerebral cortex of newborn (5-day-old) and adult rats (90-day-old). To this aim, we reexamined the heterogeneity of cortical GABA A receptors in self- and cross-competition binding experiments using [ 3H]flunitrazepam and two ligands with higher affinity for benzodiazepine BZ 1 sites relative to benzodiazepine BZ 2 sites, the triazolopyridazine 3-methyl-6-[3-(trifluoromethyl)phenyl]-1,2,4-triazolo [4,3-b] pyridazine (CL 218,872) and the imidazopyridine N, N,6-trimethyl-2-(4- methylphenyl)-imidazo[1,2-a-pyridine-3-acetemide hemitratrate (zolpidem). Benzodiazepine BZ 1 sites accounted for 52% of the total number of binding sites in adult rats, but were not detected in newborn rats. On the other hand, two classes of benzodiazepine BZ 2 sites with high and low affinity for zolpidem were present in newborn and adult rats. These sites were designated as benzodiazepine BZ 2H (high affinity for zolpidem, K d ∼ 150 nM) and benzodiazepine BZ 2L (low affinity for zolpidem, K d ∼ 3000 nM). High densities of benzodiazepine BZ 2H sites were measured in both newborn and adult rats (75% and 41% of the total number of [ 3H]flunitrazepam binding sites, respectively), whereas benzodiazepine BZ 2L sites accounted for 25% and 7% of the total number of cortical sites in neonates and adults, respectively. Flunitrazepam, CL 218,872 and zolpidem inhibited in a concentration-dependent manner the binding of [ 35 S]t- butylbicyclophosphorothionate ([ 35S]TBPS) to the convulsant site of cortical GABA A receptors in newborn and adult rats. The IC 50 for flunitrazepam was about 3-fold greater in adults than in neonates. This rightward shift in the concentration-response curve may be due to a decrease with age in the intrinsic efficacy of flunitrazepam. In contrast, CL 218,872 and zolpidem were 4-fold more potent at inhibiting [ 35S]TBPS binding in adult rats relative to neonates. The different affinities of CL 218,872 and zolpidem for benzodiazepine BZ 1 and BZ 2 receptors may account, at least in part, for the age-related changes in their inhibitory potencies. These results demonstrated that benzodiazepine BZ 2 sites mediate the modulation of [ 35S]TBPS binding by benzodiazepine recognition site ligands in the cerebral cortex of newborn rats. Further, benzodiazepine BZ 2 sites may be involved in the inhibition of [ 35S]TBPS binding by flunitrazepam, CL 218,872 and zolpidem in the cerebral cortex of adult rats.