Calcium-binding protein (CaBP) has been localized with the immunoperoxidase method using antiserum against purified chick duodenal CaBP. Different preparative procedures were employed to investigate the experimental conditions possibly responsible for the contradictory reports in the literature of the precise cellular localization of CaBP. Freeze substitution, frozen sections followed by fixation and coagulant and non-coagulant fixatives were used with appropriate control sections to demonstrate that the true localization of CaBP in the chick duodenum is in the absorptive cell cytoplasm. The goblet cell localization reported in the literature seems to be a diffusion artifact due to inadequate fixation. CaBP was also localized in several other tissues. In the hen uterus, the tubular glands beneath the surface epithelium showed intense reaction. In the kidney, CaBP was present in the cells of the straight and convoluted segments of distal tubules. The cortex of the chick cerebellum showed the CaBP in Purkinje cells. The entire dendritic trees contained the reaction product. No other neurons in the molecular or the granular layer were stained. In the deep cerebellar nuclei, all neurons were negative and these were outlined by deeply staining axons of the Purkinje cells and their synaptic endings.