The subcellular distribution of three casein kinase I (CK1) homologs, encoded by the YCK1, YCK2, and HRR25 genes, has been determined in budding yeast through a combination of subcellular fractionation and immunofluorescence methods. Both Yck proteins are tightly associated with the plasma membrane or underlying cytoskeleton and require both high-salt and nonionic detergent for extraction. Association is mediated primarily by the prenylation motif found at the C terminus of both Yck proteins. In contrast, the third CK1 homolog, Hrr25p, is found predominantly in the nucleus and only partially in the plasma membrane. Despite partial colocalization with the Yck proteins, Hrr25p is unable to rescue the yck1 delta yck2 delta phenotype. However, a chimeric kinase containing the N-terminal kinase domain of Hrr25p and the C-terminal region of Yck2p contains full Yck activity in vivo. These data suggest that members of the casein kinase I family have distinct but partially overlapping distributions in the cell that are mediated by their unique C-terminal regions.