The monoclonal antibody Cat-301 was used to examine neurons in the cerebral cortex and dorsal thalamus of several mammalian species, including Old World monkeys, cats, bush babies, guinea pigs, and rats. In each species, subpopulations of cortical and thalamic neurons are stained along the surfaces of their somata and proximal dendrites. Cat-301-positive cortical neurons include specific groups of pyramidal cells (e.g., corticospinal but not corticobulbar or callosal neurons in the monkey sensory-motor areas) and certain GABA-immunoreactive nonpyramidal cells. In the thalamus, the relay neurons projecting to the cortex and not the intrinsic neurons are stained. The Cat-301-positive neurons are nonhomogeneously distributed in the cat and monkey cortex and thalamus. In the cortex, they are densely packed in 2 bands that in most areas include layers III and V, but that in primary sensory areas include layers IV and VI. Because the density of stained neurons, their distribution, and the intensity of their staining vary among cortical areas, the borders between neighboring areas can often be detected by the differences in Cat-301 staining. Broader, regional differences are also readily apparent, for areas in the parietal and occipital lobes contain large numbers of intensely stained cells, but most areas in the frontal and temporal lobes contain fewer, more lightly stained neurons. The same broad differences are seen within the thalamus: only those nuclei reciprocally connected with intensely stained cortical areas contain large numbers of Cat-301-positive neurons. Differences among species include variations in cell density and distribution when a given cortical area or thalamic nucleus is compared between cats and monkeys. Greater differences are seen among the other species. Immunoreactive neurons in the cerebral cortex are sparse and lightly stained in guinea pigs, are restricted to the hippocampal formation in rats, and are very rare and isolated in bush babies. Similarly, Cat-301-positive thalamic neurons are restricted to only one or 2 nuclei in the guinea pig and rat and are extremely rare in the bush baby. Cat-301 stains organized groups of neurons in the cat and monkey cortex and thalamus. In addition to the laminar organization of stained cells in all cortical areas (see above), the Cat-301-positive neurons of monkey areas 17 and 18 are grouped into radial arrays. In area 17, clusters of stained cells are present in layers above and below layer IVC. These clusters lie at the centers of ocular dominance columns, within patches stained for cytochrome oxidase (CO). Most of these cells are also GABA-immunoreactive.(ABSTRACT TRUNCATED AT 400 WORDS)