Diazepam increased the acetylcholine content in the striatum and the hippocampus of the rat. This effect was antagonized in both brain areas by treatment with the specific central benzodiazepine blockers ethyl-beta-carboline-3-carboxylate and Ro 15-1788, whereas the peripheral antagonist Ro 5-4864 was ineffective. Pretreatment with picrotoxin, a known GABA antagonist did not interfere with the diazepam-induced acetylcholine increase. These results indicate a specific involvement of benzodiazepine receptors in the cholinergic action of diazepam and this effect appears to be independent of GABA receptor activation.