The present study investigates the mechanism of mouse pancreatic acinar cell apoptosis induced by H₂S in an in vitro system, using isolated pancreatic acini. Treatment of pancreatic acini with 10 µM NaHS (a donor of H₂S) for 3 hours caused phosphatidylserine externalization as shown by annexin V binding, an indicator of early stages of apoptosis. This treatment also resulted in the activation of the caspase cascade and major changes at the mitochondrial level. Caspase 3, 8 and 9 activities were stimulated by H₂S treatment. Treatment with inhibitors of caspase 3, 8 and 9 significantly inhibited H₂S-induced phosphatidylserine externalization as shown by reduced annexin V staining. The mitochondrial membrane potential was collapsed in H₂S treated acini as evidenced by fluorescence microscopy and quantitative analysis. Furthermore, the treatment of acini with H₂S caused the release of cytochrome C by the mitochondria. To investigate the mechanism underlying pancreatic acinar cell apoptosis, we also characterized the protein expression of a range of molecules that are each known to influence the apoptotic pathway. Among pro-apoptotic proteins, Bax expression is activated in H₂S treated cells but not Bid, and the anti-apoptotic proteins Bcl-X_L and Bcl-2 did not show any activation in the pancreatic acinar cell apoptosis. The death effector domain (DED) containing protein Flip is down regulated in H₂S treated acini. These results demonstrate the induction of pancreatic acinar cell apoptosis in vitro by H₂S and the involvement of both mitochondrial and death receptor pathway in the process of apoptosis.