Localization of gene expression of calbindin, a cytosolic calcium-binding protein, was examined throughout the adult rat brain by in situ hybridization with cDNA probes. The gene was expressed most intensely in the Purkinje cells in the cerebellum, intensely in the granule cells of the dentate gyrus, and moderately in the inferior olivary nucleus, in the nuclei of the trapezoid body, in the medial part of the lateral habenular nuclei, entorhinal cortex and in the mammillary nuclei. In addition, weak expression of the gene was widespread in the forebrain and brainstem gray matter, and also in small cells in the spinal posterior horn as well as the ependymal cells. The widespread and heterogeneous expression of the gene in the brain suggests that calbindin is differentially involved in calcium-regulated phenomena in different neurons.