Recent studies report the Src-family kinases (SFK's) are important in a number of physiological and pathophysiological responses of pancreatic acinar cells (pancreatitis, growth, apoptosis); however, the role of SFKs in various signaling cascades important in mediating these cell functions is either not investigated or unclear. To address this we investigated the action of SFKs in these signaling cascades in rat pancreatic acini by modulating SFK activity using three methods: adenovirus-induced expression of an inactive dominant-negative CSK (Dn-CSK-Advirus) or wild-type CSK (Wt-CSK-Advirus), which activate or inhibit SFK, respectively, or using the chemical inhibitor, PP2, with its inactive control, PP3. CCK (0.3, 100 nM) and TPA (1 μM) activated SFK and altered the activation of FAK proteins (PYK2, p125(FAK)), adaptor proteins (p130(CAS), paxillin), MAPK (p42/44, JNK, p38), Shc, PKC (PKD, MARCKS), Akt but not GSK3-β. Changes in SFK activity by using the three methods of altering SFK activity affected CCK/TPAs activation of SFK, PYK2, p125(FAK), p130(CAS), Shc, paxillin, Akt but not p42/44, JNK, p38, PKC (PKD, MARCKS) or GSK3-β. With chemical inhibition the active SFK inhibitor, PP2, but not the inactive control analogue, PP3, showed these effects. For all stimulated changes pre-incubation with both adenoviruses showed similar effects to chemical inhibition of SFK activity. In conclusion, using three different approaches to altering Src activity allowed us to define fully for the first time the roles of SFKs in acinar cell signaling. Our results show that in pancreatic acinar cells, SFKs play a much wider role than previously reported in activating a number of important cellular signaling cascades shown to be important in mediating both acinar cell physiological and pathophysiological responses.