Whilst the brain-specific benzodiazepine receptor has been assumed to show a late evolutionary appearance, we present evidence for the presence of a central benzodiazepine binding site in sharks, which shows a high affinity for [ 3H]Ro 15-1788. However, the receptor density and the affinities of several benzodiazepine receptor ligands are lower than in mammals, thus presumably explaining why the benzodiazepine binding sites had previously escaped detection in elasmobranchs. Additionally, radio- and immunohistochemistry were performed to localize the radioligand binding sites and the antigenic sites of the shark γ-aminobutyric acid (GABA)-benzodiazepine receptor. In cerebellum, the granular layer reveals a high density of [ 3H]muscimol binding sites. The immunoreaction obtained with the β-subunit-specific monoclonal antibody bd-17 seemingly parallels the distribution of high-affinity GABA binding sites. In contrast, [ 3H]Ro 15-1788 binding sites are evenly distributed in the molecular and granular layers, thus the results are similar to those previously described for rat cerebellum. Apparently, the respective distributions in this brain region are well conserved throughout vertebrate evolution.