The tissue distribution, developmental changes, and the vitamin D dependence in the rat of two calcium-binding proteins [CaBPs, 28,000 and 10,000 Mr (28 and 10 K)] were examined. The radioimmunoassays used employed specific antibodies to either the human cerebellar CaBP (28 K protein) or to the smaller rat intestinal CaBP (10 K protein). The assay for the 28 K CaBP may be used to detect this protein in a number of mammalian species and tissues, whereas the 10 K CaBP assay appears to be specific only for the rat intestinal CaBP. This report demonstrates that the tissue distribution of the two CaBPs is different in the rat. High levels of the 28 H protein were found in the cerebellum and kidney, whereas the smaller CaBP was concentrated in the duodenum, jejunum, and cecum. Many other organs and tissues contained small quantities of both CaBPs. Developmental studies indicated some variability in the concentration of the CaBPs. Duodenum, kidney, and cerebellum all contained small amounts of one of the CaBPs prior to birth. Adult levels in all three tissues were already reached at 30 postpartum days. Levels of both CaBPs began to decline in rats older than 2 mo. The vitamin D dependence appeared to reflect cell turnover in that the duodenal and kidney CaBPs showed a vitamin D dependence not observed for the cerebellar protein.