The heat shock protein 70 family members Hsc70 and Hsp70 are known to play a protective role against the onset of experimental pancreatitis, yet their molecular function in acini is unclear. Cysteine string protein (CSP) is a zymogen granule (ZG) membrane protein characterized by an N-terminal 'J domain' and a central palmitoylated string of cysteine residues. The J domain functions as a cochaperone by modulating the activity of Hsc70/Hsp70 family members. A role for CSP in regulating digestive enzyme exocytosis from pancreas was investigated by introducing CSP truncations into isolated acini following their permeabilization with Perfringolysin O. Incubation of acini with CSP_1-82, containing the J domain, significantly augmented Ca²⁺-stimulated amylase secretion. Effects of CSP_1-82 were concentration-dependent, with a maximum 80% increase occurring at 200 µg/ml of protein. Although CSP_1-82 had no effects on basal secretion measured in the presence of 10 nM free Ca²⁺, it did significantly augment GTPS-induced secretion under basal Ca²⁺ conditions by approximately 25%. Mutation of the J domain to abolish its cochaperone Hsc70 failed to augment Ca²⁺-stimulated secretion implicating the CSP/Hsc70 cochaperone system as a regulatory component of the secretory pathway. CSP physically associates with vesicle associated membrane protein 8 (VAMP 8) on ZGs and the CSP-VAMP 8 interaction was dependent on amino acids 83-112 of CSP. Immunofluorescence analysis of acinar lobules or purified ZGs confirmed the CSP colocalization with VAMP 8. These data establish a role for CSP in regulating digestive enzyme secretion and suggest that CSP and Hsc70 modulate specific SNARE interactions necessary for exocytosis.