In order to test the hypothesis emerging from the literature that 3H-sulpiride and 3H-spiperone might label different subclasses of dopamine D2 receptors in the brain, the binding properties of the two ligands were compared under optimized conditions. 3H-sulpiride was found to show a very fast rate of dissociation from the receptor. Furthermore, with this ligand, an apparently "specific" binding to glass fiber filters was observed. A centrifugation assay was therefore used for the characterization of 3H-sulpiride binding. The binding of 3H-spiperone was measured in a way which prevented its attachment to sites other than D2 receptors. Under these conditions, the two ligands were found to label identical dopaminergic recognition sites (D2 receptors) according to the following criteria: 3H-sulpiride and 3H-spiperone both labelled only one single site with a comparable density. Identical changes in the receptor number for both ligands were found in the tissue from animals with nigrostriatal 6-hydroxydopamine lesions. The potencies of various dopamine agonists and antagonists in displacing 3H-sulpiride or 3H-spiperone from their binding sites showed a highly significant one to one correlation. Finally, the binding of both ligands could be inactivated by pretreatment of the membranes with N-ethylmaleimide, with or without Na+-ions being present. This inactivation followed exactly the same kinetics for both radioligands. Thus, no indication supporting the hypothesis that 3H-sulpiride and 3H-spiperone might label different subsets of dopamine D2 receptors in the rat brain could be found.