Mammalian casein kinase 1delta (CK1d) is a homologue of the S. cerevisiae Hrr25p protein kinase. Hrr25p is involved in regulating diverse events including vesicular trafficking, gene expression, DNA repair, and chromosome segregation. In contrast to Hrr25p, little is known about the function, regulation, or subcellular localization of CK1d. In the present study, we show that CK1d in mammalian cells is mainly cytoplasmic and enriched within the Golgi and/or ER-Golgi transport vesicles, consistent with a role in vesicular trafficking. Transient expression of green fluorescent protein (GFP)- or FLAG peptide-tagged CK1d showed localization similar to that of the endogenous CK1d. GFP-CK1d was also enriched at the centrosomes in interphase cells. Strikingly, two inactive mutant CK1dproteins (K38M and T176I) showed almost exclusive nuclear staining, suggesting that protein kinase activity is required for normal localization of CK1d and prevention of nuclear accumulation. The nuclear export inhibitor leptomycin B promoted nuclear enrichment of CK1d indicating that nuclear localization of CK1d occurs physiologically. Both endogenous CK1d and GFP-CK1delta are enriched on the spindle poles in mitotic cells, consistent with a role in regulating spindle formation. Localization is a property of the protein kinase domain and is independent of the C-terminal noncatalytic domain. These data are consistent with roles for CK1d in mammalian cells analogous to those of its yeast counterparts.