The calcium channel antagonists omega-agatoxin IIIA (omega-Aga-IIIA) and omega-conotoxin GVIA (omega-CgTx) were radioiodinated and used to locate binding sites in the rat brain by receptor autoradiography. While patterns of regional binding to sagittal sections of rat brain were generally similar for the 2 toxins, notable differences in the cerebellum and hippocampus were observed. Specific [125I]omega-Aga-IIIA binding was greatest in the granule cell layers of the cerebellum and of the dentate gyrus. In contrast, binding of [125I]omega-CgTx was most intense in the molecular layers of these structures. Less than one-third of [125I]omega-Aga-IIIA binding in rat brain slices was inhibited by pre-exposure to 250 nM omega-CgTx, while 40 nM omega-Aga-IIIA virtually eliminated the binding of [125I]omega-CgTx under the same conditions. The P-type calcium channel antagonist omega-Aga-IVA blocked only a small fraction of [125I]omega-Aga-IIIA and [125I]omega-CgTx binding. These autoradiographic data are consistent with membrane binding experiments and indicate that the combined use of agatoxins and conotoxins may be useful in the characterization of separate types of neuronal calcium channels.