CRHSP-24 is a serine-phosphoprotein originally identified as a physiological substrate for the Ca²⁺/calmodulin regulated protein phosphatase calcineurin (also known as PP2B). CRHSP-24 is a paralog of the brain specific mRNA binding protein PIPPIN and was recently shown to interact with the STYX/dead-phosphatase protein in developing spermatids (Wishart and Dixon, PNAS 99; 2112-2117). Investigation of the effects of phorbol ester (TPA) and cAMP analogs in ³²P-labeled pancreatic acini revealed these agents acutely dephosphorylated CRHSP-24 by a Ca²⁺-independent mechanism. Indeed, the cAMP- and TPA-mediated dephosphorylation of CRHSP-24 was fully inhibited by the PP1/PP2A inhibitor calyculin A indicating the protein is regulated by an additional phosphatase other than PP2B. Supporting this, CRHSP-24 dephosphorylation in response to the Ca²⁺-mobilizing hormone cholecystokinin was differentially inhibited by calyculin A and the PP2B selective inhibitor cyclosporin A. Stimulation of acini with secretin, a secretagogue that signals through the cAMP pathway in acini, induced CRHSP-24 dephosphorylation in a concentration dependent manner. Isoelectric focusing and immunoblotting indicated that elevated cellular Ca²⁺ dephosphorylated CRHSP-24 on at least 3 serine sites, whereas, cAMP and TPA partially dephosphorylated the protein on at least 2 sites. The cAMP-mediated dephosphorylation of CRHSP-24 was inhibited by low concentrations of okadaic acid (10 nM) and fostriecin (1 µM) suggesting CRHSP-24 is regulated by PP2A or PP4. Collectively, these data indicate that CRHSP-24 is regulated by diverse and physiologically relevant signaling pathways in acinar cells including Ca²⁺, cAMP and diacylglycerol.