The distribution of adenosine A1 receptors was studied quantitatively in the brain of the rat, mouse, guinea-pig and cat, using in vitro autoradiography with [3H]N6-cyclohexyladenosine as ligand. Preliminary binding studies in brain sections from the guinea-pig and cat gave results similar to previous data from the rat and showed that the binding site had the pharmacological profile of an A1 receptor. The overall distribution of receptors was comparable in the species studied. The receptors were concentrated in the hippocampus, the cerebral cortex, some thalamic nuclei, the basal ganglia and the cerebellar cortex. The hypothalamus and the brainstem were sparse in receptors. Differences among species in receptor distribution and/or density were seen in some regions, e.g. the cerebral cortex, the striatum, the lateral geniculate nucleus and the cerebellar cortex. The autoradiograms were compared with adjacent sections stained for 5'-nucleotidase. There was in general a poor correlation between the distribution of A1 receptors and 5'-nucleotidase. Furthermore, there were marked differences between species in the distribution of the enzyme. The species differences observed in receptor localization may be of functional relevance.