Tumor protein D52 (also known as CRHSP-28) is highly expressed in multiple cancers and tumor-derived cell lines; however, it is normally abundant in secretory epithelia throughout the digestive system, where it has been implicated in Ca(2+)-dependent digestive enzyme secretion (41). Here we demonstrate, using site-specific mutations, that Ca(2+)-sensitive phosphorylation at serine 136 modulates the accumulation of D52 at the plasma membrane within 2 min of cell stimulation. When expressed in Chinese hamster ovary CHO-K1 cells, D52 colocalized with adaptor protein AP-3, Rab27A, vesicle-associated membrane protein VAMP7, and lysosomal-associated membrane protein LAMP1, all of which are present in lysosome-like secretory organelles. Overexpression of D52 resulted in a marked accumulation of LAMP1 on the plasma membrane that was further enhanced following elevation of cellular Ca(2+). Strikingly, mutation of serine 136 to alanine abolished the Ca(2+)-stimulated accumulation of LAMP1 at the plasma membrane whereas phosphomimetic mutants constitutively induced LAMP1 plasma membrane accumulation independent of elevated Ca(2+). Identical results were obtained for endogenous D52 in normal rat kidney and HeLA cells, where both LAMP1 and D52 rapidly accumulated on the plasma membrane in response to elevated cellular Ca(2+). Finally, D52 induced the uptake of LAMP1 antibodies from the cell surface in accordance with both the level of D52 expression and phosphorylation at serine 136 demonstrating that D52 altered the plasma membrane recycling of LAMP1-associated secretory vesicles. These findings implicate both D52 expression and Ca(2+)-dependent phosphorylation at serine 136 in lysosomal membrane trafficking to and from the plasma membrane providing a novel Ca(2+)-sensitive pathway modulating the lysosome-like secretory pathway.