CRHSP-28 is a member of the tumor-protein D52 (TPD52) family that is highly expressed in exocrine glands and was recently shown to regulate digestive enzyme secretion from pancreatic acinar cells. The current study reveals that CRHSP-28 is highly expressed in the cultured mucosal secretory cell line T84, findings consistent with an important regulatory role for the protein in apical membrane trafficking. Stimulation of cells with the muscarinic cholinergic agonist carbachol (CCh) induced a rapid, concentration-dependent phosphorylation of CRHSP-28 on at least 2 serine residues. Isoelectric focusing and immunoblotting were used to characterize the cellular mechanisms governing CRHSP-28 phosphorylation. Phosphorylation is dependent on elevated cellular Ca²⁺ as it was maximally induced by the Ca²⁺ ionophore ionomycin and the endoplasmic reticulum Ca²⁺-ATPase inhibitor thapsigargin, and fully inhibited by the intracellular Ca²⁺ buffer BAPTA-AM. In agreement with the presence of 3 consensus sites for serine phosphorylation by casein kinase II (CKII), recombinant CRHSP-28 was phosphorylated in vitro to a 10-fold greater extent by CKII than Ca²⁺/calmodulin-dependent protein kinase II (CaMKII), for which no obvious consensus site (arg-X-X-ser) is present in the protein. However, phospho-peptide mapping studies demonstrated that CaMKII induced an identical phospho-peptide profile as that seen for endogenous CRHSP-28 immunoprecipitated from T84 cells. Although calmodulin antagonists had no effect on CCh-stimulated CRHSP-28 phopshorylation, disruption of actin filaments by cytochalasin D inhibited phosphorylation by 50%. Confocal microscopy indicated CRHSP-28 is expressed in perinuclear regions of cells and accumulates immediately below the apical membrane of polarized monolayers following CCh stimulation. CaM kinase II was also localized to the sub-apical cytoplasm, and was clearly displaced from this region following actin-filament disruption. Collectively, these data suggest that CRHSP-28 phopshorylation is regulated by a CaMKII-like enzyme and likely involves a translocation of the protein within the apical cytoplasm of epithelial cells.