The cholecalcins are intracellular vitamin D-dependent calcium-binding proteins. High concentrations of 28 kDa cholecalcins have been found in the kidneys and cerebella of birds and mammals. However, whereas the synthesis of the renal protein is vitamin D-dependent, that of the cholecalcin in the cerebella of young growing chicks and rats is apparently not. In the present study a range of immunological, physicochemical and structural characteristics of renal and cerebellar cholecalcins isolated from a single species, the rat, has been examined to ascertain what, if any, differences there are between them, other than the vitamin D-dependence. Both proteins behaved in exactly the same way during purification and showed complete immunocross-reactivity in Ouchterlony double immunodiffusion and radioimmunoassay. No difference could be detected between them on electrophoresis under denaturing conditions or on two-dimensional isoelectric focusing/electrophoresis. Their amino acid compositions were very similar, as were their u.v.-absorption spectra and their chymotryptic and tryptic peptide maps. The N-terminal sequence was found to be Gly-Gly-Val-Ser... for both cholecalcins. We have thus been unable to show any significant difference between the two proteins and suggest that the 28 kDa cholecalcins of the rat cerebellum and kidney are extremely similar, if not identical.