In this study we have employed a synthetic peptide of the rab3a effector domain, rab3AL, to examine whether a rab-like low molecular weight GTP-binding protein is involved in protein release from the rat pancreatic acinar cell. The peptide was found to be a potent stimulator of amylase release from streptolysin-O-permeabilized pancreatic acini, with an EC50 of approximately 60 microM. Stimulation of amylase discharge by rab3AL did not occur using either intact acini or permeabilized acini depleted of ATP. In contrast, a different effector domain peptide of the rab2 protein, rab2AL, a peptide with distinct sequence homology to rab3AL, was unable to stimulate amylase release, suggesting the specificity of the rab3AL response to rab3-like proteins. rab3AL stimulated release at [Ca2+] that were nonstimulatory in the absence of the peptide (10 nM). rab3AL potentiated the effect of guanosine 5'-[gamma-thio]triphosphate on amylase secretion and decreased the amount of guanosine 5'-[gamma-thio]triphosphate required for maximal secretion, suggesting that these two agents interact to modulate a distal step(s) of secretion. The above results provide functional evidence for the role of a rab-like low molecular weight GTP-binding protein and its effector protein(s) in the control of protein release from pancreatic acini. Because the discharge response to rab3AL is near the maximal obtainable from permeabilized acini, our results would suggest that rab3-like proteins control an important step in regulated secretion of amylase.