Mechanisms modulating the pituitary adenylate cyclase activating polypeptide (PACAP)-induced increase in excitability have been studied using dissociated guinea pig intrinsic cardiac neurons and intact ganglion preparations. Measurements of intracellular calcium (Ca2+) with the fluorescent Ca2+ indicator dye fluo-3 indicated that neither PACAP nor vasoactive intestinal polypeptide (VIP) at either 100 nM or 1 microM produced a discernible elevation of intracellular Ca2+ in dissociated intracardiac neurons. For neurons in ganglion whole mount preparations kept in control bath solution, local application of PACAP significantly increased excitability, as indicated by the number of action potentials generated by long depolarizing current pulses. However, in a Ca2+ -deficient solution in which external Ca2+ was replaced by Mg2+ or when cells were bathed in control solution containing 200 microM Cd2+, PACAP did not enhance action potential firing. In contrast, in a Ca2+ -deficient solution with Ca2+ replaced by strontium (Sr2+), PACAP increased excitability. PACAP increased excitability in cells treated with a combination of 20 microM ryanodine and 10 mM caffeine to interrupt release of Ca2+ from internal stores. Experiments using fluo-3 showed that ryanodine/caffeine pretreatment eliminated subsequent caffeine-induced Ca2+ release from intracellular stores, whereas exposure to the Ca2+ -deficient solution did not. In dissociated intracardiac neurons voltage clamped with the perforated patch recording technique, 100 nM PACAP decreased the voltage-dependent barium current (IBa). These results show that, in the guinea pig intracardiac neurons, the PACAP-induced increase in excitability apparently requires Ca2+ influx through Cd2+ -sensitive calcium permeable channels other than voltage-dependent Ca2+ channels, but not Ca2+ release from internal stores.