In order to clarify the mechanism by which pGlu-Asn-Cys(Cys)-Pro-Arg-Gly-NH(2) (vasopressin-(4-9)), a major metabolite C-terminal fragment of [Arg(8)]-vasopressin (vasopressin-(1-9)), improves learning and memory, we used several different drugs such as an acetylcholine receptor antagonist, a Ca(2+)/calmodulin-dependent protein kinase II inhibitor, vasopressin receptor antagonists and L-type Ca(2+) channel blocker to disrupt spatial memory in rats. Moreover, we examined the effect of vasopressin-(4-9) on acetylcholine release in the ventral hippocampus using microdialysis. Vasopressin-(4-9) (10 fg/brain, i.c.v.) improved the impairment of spatial memory in the eight-arm radial maze induced by scopolamine, pirenzepine and Ca(2+)/calmodulin -dependent protein kinase II inhibitor. Pirenzepine, a vasopressin V(1A) receptor antagonist, and L-type Ca(2+) channel blocker, but not a vasopressin V(2) receptor antagonist, suppressed the effects of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory. Moreover, vasopressin-(4-9) did not affect acetylcholine release in the ventral hippocampus of intact rats or of scopolamine-treated rats as assessed by microdialysis. These results suggest that vasopressin-(4-9) activates vasopressin V(1A) receptors on the postsynaptic membrane of cholinergic neurons, and induces a transient influx of intracellular Ca(2+) through L-type Ca(2+) channels to interact with muscarinic M(1) receptors. The activation of these processes by vasopressin-(4-9) is critically involved in the positive effect of vasopressin-(4-9) on scopolamine-induced impairment of spatial memory.