This study examined and compared the immunocytochemical distribution of the two calcium-binding proteins parvalbumin and calbindin-D28k in the primary motor and somatosensory areas of the rat neocortex. Parvalbumin-immunoreactive cells were found in all layers of the cortex except layer 1 and reached their peak density in the middle layers. The two cortical areas differed markedly in the number, cell size and morphology of immunoreactive cells. Parvalbumin-positive cells were more than twice as numerous in the somatosensory cortex compared to the motor cortex. In addition, the average size of their cell bodies was 25-30% larger in the somatosensory area. Parvalbumin cells in the motor area represented several classes of nonpyramidal cells, while the somatosensory cortex contained in addition many large cells with thick vertically oriented primary dendrites. Some of these cells resembled regular or inverted pyramidal neurons. Punctate neuropil labeling was much heavier in the upper layers of the somatosensory than in the motor cortex and was especially heavy in layer 4. Dense parvalbumin-positive perisomatic puncta surrounded large, unstained pyramidal cells in layer 5B of the motor cortex. Calbindin-D28k neuronal staining in both areas was confined to two populations. The most prominent was darkly labeled, small nonpyramidal cells confined to two bands in layers 2/3 and 5/6. There was also a lighter stained population composed of many pyramidal cells distributed throughout layers 2 and 3. In addition, the motor area contained a band of lightly stained, large pyramidal cells in layer 5B. Calbindin-D28k neuropil labeling was heaviest in layers 1 to 3. In contrast to parvalbumin, we found only minor differences in distribution, size and morphology of calbindin-D28k cell body or neuropil staining in the two cortical areas. Double-labeling immunocytochemistry showed that the large majority of immunoreactive cells contained only calbindin-D28k or parvalbumin, but a distinct population of multipolar cells in the upper layers of the somatosensory cortex contained both. The clear parcellation of parvalbumin immunoreactivity in the rat neocortex suggests that parvalbumin is preferentially associated with specific neuronal populations and terminals in the somatosensory cortex. The more general and homogeneous labeling of the upper layers of the cortex indicates that calbindin-D28k could be related to the relatively high density of calcium channels or N-methyl-D-aspartate receptors in the superficial layers of the rat cortex.