After the discovery of calretinin, a protein with high sequence homology to calbindin D-28k, the validity of immunohistochemical results obtained using polyclonal antibodies for this protein, was in question. In order to validate the previous results on the localization of calbindin D-28k in the brain, we localized the protein by highly specific monoclonal antibodies and revealed its mRNA histochemically by in situ hybridization. In general there was good agreement between the results obtained using these two different techniques and those reported in previous publications. The concordance was particularly impressive for the cerebral cortex, basal ganglia, basal nucleus of Meynert, hippocampus, thalamus, cerebellum and superior colliculus. In the amygdala and hypothalamus the low spatial resolution of in situ hybridization did not allow precise definition of some nuclei displaying a positive reaction for the protein. In the rhombencephalon, cells of the parabrachial nuclei and the dorsal raphe nucleus expressed calbindin D-28k. Neurons in the dorsal horn of the spinal cord and some horizontal cells of the retina were tagged with both methods. The only discrepancy was the presence of immunoreactive ependymal cells, whereas mRNA never occurred in cells lining the ventricles. Thus, the combined approach has established the widespread distribution of cells expressing calbindin D-28k in the rat brain.