The role of hippocampus in the anxiolytic-like effect of buspirone in the conditioned emotional response test (CER, a freezing response), was examined by immunocytochemical detection of the c-Fos protein. It was shown that buspirone at the dose of 0.5 and 1.5 mg/kg i.p. given before test session, which was 24 h after the aversive training, significantly decreased freezing response within a limited dose range of the U-shaped dose–response relationship. Exposure of animals to aversively conditioned context (a contextual fear) induced the production of c-Fos protein in the dentate gyrus, CA-1 and CA-3 layers of the hippocampus. Pretreatment with buspirone (1.5 mg/kg) significantly attenuated the effects of aversive memory on c-Fos protein expression in the CA-1 and CA-3 layers of the hippocampus. These immunocytochemical results support previous data obtained in our laboratory with the help of selective neurotoxic lesions and intrahippocampal drug injections suggesting an important role of hippocampus in the anxiolytic effects of buspirone.