Recent reports have shown that there exists in mammalian brain a number of heat-stable Ca2(+)-binding proteins that are distinct from calmodulin [McDonald & Walsh (1985) Biochem. J. 232, 559-567]. We have attempted to characterize equivalent Ca2(+)-binding proteins from Drosophila. Affigel-phenothiazine chromatography, which can be used to purify calmodulin and other Ca2(+)-binding proteins, allowed the identification of a possible heat-stable 23 kDa Ca2(+)-binding protein. A purification procedure for this protein has been devised. Purified 23 kDa protein shows characteristics typical of a Ca2(+)-binding protein; there is a mobility shift on SDS/polyacrylamide gels in the presence of EGTA, and Western blotting, followed by the use of the 45Ca2+ overlay technique, confirms that the 23 kDa protein does bind Ca2+. 45Ca2+ binding studies indicate that this protein binds 1 mol of Ca2+/mol of protein, with Kd 1.9 microM. A single band with pI 5.2 is obtained on isoelectric focusing. Analysis of Western blots of Drosophila tissues probed with antibodies to the Ca2(+)-binding protein indicates that it has a widespread distribution, but is absent from muscle tissue. The antibodies also cross-react with a protein of identical molecular mass in extracts of sheep brain. The possible similarity between this Drosophila Ca2(+)-binding protein and mammalian proteins is discussed, and comparison is made between this Drosophila protein and other Ca2(+)-binding proteins purified from vertebrates.