Tumor protein D52 is expressed at relatively high levels in cells within the GI tract that undergo classical exocytosis and is overexpressed in several cancers. Current evidence supports a role for D52 in the regulation of vesicular trafficking. D52 function(s) are regulated by calcium-dependent phosphorylation; however, the intracellular mechanisms that mediate this process are not well characterized. The goal of this study was to identify the calcium-dependent phosphorylation site(s) in D52 and to characterize the protein kinase(s) that mediate this phosphorylation. Using mass spectrometry and site-directed mutagenesis, we identified a single amino acid residue, S¹³⁶, that undergoes increased phosphorylation upon elevation of [Ca²⁺]i. A phosphospecific antibody (pS¹³⁶) was produced and used to characterize D52 kinase activity in gastric mucosal, colonic T84 and HEK293 cells. Using D52 as a substrate, a protein kinase with a molecular weight (Mr) of ~50 kDa was identified with "in gel" assays. This kinase co-migrated with rat brain calcium/calmodulin-dependent protein kinase (CAMK2), cross-reacted with pan-specific CAMK2 antibodies as well as with anti-active CAMK2 (pT^286/287) antibody when activated. Carbachol-stimulated phosphorylation of S136 was inhibited by the CAMK2 inhibitor, KN93 (IC50, 38 µM), and by the calmodulin antagonist, W7 (IC50, 3.3 nM). A previously uncharacterized CAMK2 isoform, CAMK26, which has the same domain structure and Mr as CAM2, was identified in gastric mucosa using RT-PCR. The cloned, expressed protein comigrated with D52 kinase and colocalized with D52 protein in T84 and HEK293 cells. These findings support a role for CAMK26 in the mediation of D52 phosphorylation.