The effects of PACAP on hepatic glucose metabolism were examined using the flow-through perfusion method for fed rat livers, because some of the glucagon superfamily peptides stimulate hepatic glucose output. Glucose output was significantly stimulated in a dose-dependent manner by more than 1 nM PACAP-27. The potency of its stimulation was equal to that of PACAP-38, greater than that of VIP, and clearly lower than that of glucagon. The cAMP output was also increased significantly by more than 1 nM PACAP-27; however, the degree and profile of cAMP output were not in parallel with those of glucose. Theophylline did not affect these stimulatory effects. On the other hand, in the perfusion experiment with Ca2+ free perfusate, the degrees of increase in glucose output induced by 15 and 40 nM PACAP-27 were significantly reduced. In conclusion, PACAP stimulates glucose output from the perfused rat liver, and Ca2+ rather than cAMP plays an important role in this action as a second messenger.