Ca²⁺-regulated heat stable protein (CRHSP-28) is a member of the TPD52 protein family that has been shown to regulate Ca²⁺-dependent secretory activity in pancreatic acinar cells. Immunofluorescence microscopy of isolated lobules demonstrated CRHSP-28 is localized to a supranuclear apical compartment in acini and accumulates immediately below the apical membrane within 2 min of CCK-8 stimulation. Dual immunofluorescence microscopy demonstrated an endosomal localization of CRHSP-28, which strongly overlapped with early endosomal antigen-1 (EEA1) on vesicular structures throughout the apical cytoplasm but showed only minimal overlap with the transferrin receptor, which is present in basolaterally derived endosomes. Significant overlapping of CRHSP-28 with the trans-Golgi network marker TGN38 was also noted in supranuclear regions of acini. Interestingly, treatment of lobules with brefeldin A reversibly disrupted the vesicular localization of both CRHSP-28 and EEA1 within the apical cytoplasm. The CCK-8 induced accumulation of CRHSP-28 in subapical regions of acini was not altered by inhibiting apical endocytosis using the actin filament disrupting agent latrunculin B. Immunoelectron microscopy confirmed CRHSP-28 is associated with the limiting membrane of irregularly shaped vesicular structures of low electron density in the apical cytoplasm that are positive for EEA1 staining. Sparse but significant CRHSP-28 immunoreactivity was also observed along the limiting membrane of zymogen granules. Consistent with immunofluorescence data, CRHSP-28 was found to accumulate in clusters on endosomes and positioned between zymogen granules below the cell apex upon CCK-8 stimulation. These data indicate that CRHSP-28 is present within both endocytic and exocytic compartments of acinar cells and is acutely regulated by secretagogue stimulation.