We report the molecular cloning and characterization of a 49-kDa form of casein kinase I from rat testis. A cDNA clone encoding the enzyme, designated casein kinase I delta, contained an open reading frame of 1284 nucleotides that predicts a polypeptide of 428 amino acids with a M(r) of 49,121. The predicted amino acid sequence shares 76% identity with casein kinase I alpha, a 37-kDa form recently cloned from bovine brain (Rowles, J., Slaughter, C., Moomaw, C., Hsu, J., and Cobb, M. (1991) Proc. Natl. Acad. Sci. U. S. A. 88, 9548-9552), and 65% identity with HRR25, a 57-kDa form of casein kinase I from yeast shown to be involved in DNA repair (Hoekstra, M. F., Liskay, R. M., Ou, A. C., DeMaggio, A. J., Burbee, D. G., and Heffron, F. (1991) Science 253, 1031-1034). Northern analysis of rat or rabbit RNA revealed three hybridizing species of 3.5-4.1, 2.2, and 1.9 kilobase pairs (kb). The largest message was detected in all tissues examined, whereas the 1.9- and 2.2-kb species were found predominantly in testis. A probe corresponding to the 3'-untranslated region of the casein kinase I delta cDNA hybridized only to the 1.9-kb transcript. Expression of the casein kinase I delta cDNA in Escherichia coli resulted in active enzyme that phosphorylated casein, phosvitin, and the peptide substrate DDDDVASLPGLRRR. Enzyme activity was associated with a predominant polypeptide of 55-kDa, although COOH-terminal degradation products of 50 and 42 kDa were also present in partially purified enzyme. Recombinant casein kinase I delta was inhibited by the specific casein kinase I inhibitor, CKI-7, half-maximally at 12 microM. Heparin inhibited recombinant casein kinase I delta when phosvitin was the substrate, with half-maximal inhibition at 11.5 micrograms/ml. However, if the peptide substrate was used, heparin activated recombinant casein kinase I delta 4-5-fold, with half-maximal activation at 9.5 micrograms/ml. A truncated form of casein kinase I delta, lacking the COOH-terminal 111 amino acids, was no longer activated by heparin. Casein kinase I delta therefore represents a separate member of the casein kinase I family distinguished by its larger size and unique kinetic behavior with respect to heparin.