Protein phosphorylation is believed to play a role in the regulation of ciliary motility in the protozoan Paramecium tetraurelia. Five protein kinases from Paramecium, activated by cyclic nucleotides or Ca2+, have been characterized previously. We report here the identification of a family of second-messenger-independent casein kinases in Paramecium. Casein kinase activity was enriched in the soluble fraction of cilia, but there was also significant activity tightly associated with axonemes. Three ciliary casein kinase activities (soluble CKS1 and CKS2, and axonemal CKA) were separated by chromatography and characterized. The native forms of all three were monomeric, with molecular masses of 28-45 kDa as judged by in-gel kinase assays and sizing by gel filtration. CKS2 was inhibited by heparin, but CKA was unaffected and CKS1 was stimulated. All three activities preferred acidic substrates such as casein and phosvitin, but they could be distinguished by their preference for other substrates. Antibodies against mammalian casein kinase I recognized CKS1 and CKS2 in immunoblots (43 kDa), but did not stain CKA. The antibodies to casein kinase I were used to probe other cellular fractions. A 65 kDa antigen (particulate casein kinase, CKP) was enriched in particulate fractions of whole cells. This 65 kDa protein was found in isolated cell cortices, but was not present in the infraciliary lattice. This report represents the first biochemical identification of a casein kinase I family in protozoa.