Pharmacological characterization of [ 3H]benzodiazepine binding to membrane preparations of adult rat hippocampus and neonatal rat brain have demonstrated, in addition to the ω 1 and ω 2 populations of central ω benzodiazepine binding sites associated with GABA A receptors, the existence of binding sites with μM affinity for the imidazopyridines zolpidem and alpidem. In the present study we have investigated their comparative autoradiographic distribution using [ 3H]flumazenil as a ligand. In the neonatal rat CNS, the imidazopyridine derivatives zolpidem and alpidem were found to discriminate two [ 3H]flumazenil binding site populations with an IC 50 value ratio of more than 200-fold. In the different regions investigated (spinal cord, striatum, neocortex and inferior colliculus) the low affinity component had IC 50 values of 20–40 μM (zolpidem) and 5–15 μM (alpidem) and accounted for ca. 50% of the total binding site population. In the adult rat, these imidazopyridine derivatives displayed a greater displacing potency in the cerebellum (IC 50 = 6 and 36 nM, respectively) than in the hippocampus (IC 50 = 37 and 403 nM, respectively). In the cerebellum, [ 3H]flumazenil binding was fully displaced by 1 μM of either compound and Hill coefficients of displacement curves were close to unity. In the hippocampus, 25% of [ 3H]flumazenil binding were resistant to 3 μM zolpidem or 1 μM alpidem, but were displaced by 100 μM of either compound. CL 218,872 also displayed a greater displacing potency in the cerebellum (IC 50 = 83 nM) than in the hippocampus (IC 50 = 711 nM) but [ 3H]flumazenil binding in the hippocampus was fully displaced by 10 μM of this compound. In adult rat hippocampus, zolpidem and alpidem were found to discriminate between three central ω site subtypes which display high (IC 50 = 31 and 6.1 nM, respectively; ω 1 sites), intermediate (IC 50 = 480 and 122 nM, respectively; ω 2I) and low (IC 50 = 20100 and 2300 nM, respectively; ω 2L) affinity for these imidazopyridine derivatives. In contrast, CL 218,872 discriminated between ω 1 and ω 2 sites but not between two ω 2 receptor subpopulations. ω 1 sites were mainly localized in layer IV of the sensorimotor cortex, cerebellum, substantia nigra, olfactory bulb and inferior collicus. ω 2I sites were present in the cortical mantle (with higher levels in the cingulate and olfactory than in the sensorimotor cortex) and in subcortical (hippocampus, hypothalamus and nucleus accumbens) limbic structures. In the hippocampus, hypothalamus, spinal cord and nucleus accumbens, ω 2L sites accounted for more than 25% of the specific [ 3H]flumazenil binding; the density of these sites was minor in the cortex and in most pyramidal and extrapyramidal system structures. In conclusion, the present results indicate that in adult and neonatal rat CNS sections zolpidem and alpidem can discriminate between 3 populations of [ 3H]flumazenil binding sites that may be akin to ω binding sites associated with GABA A receptors containing α 1-subunits (ω 1), α 2- or α 3-subunits (ω 2I) and α 5-subunits (ω 2L, respectively.