This study was done to determine if pituitary adenylate cyclase-activating peptide (PACAP)-immunoreactive nerve fibers occur in cardiac muscle as well as intracardiac ganglia of rats and guinea pigs and to clarify the chronotropic actions of PACAP27 in the same species using isolated heart preparations. PACAP nerve fibers were not detected in atrial or ventricular muscle of rat or guinea pig but a few stained nerve fibers occurred in the atrioventricular bundle of the guinea pig. Stained nerve fibers were prominent in intracardiac ganglia of both species. PACAP27 caused a dose-dependent tachycardia in isolated rat hearts (+39 +/- 3 beats/min with 1 nmol, n = 6). Positive and/or negative chronotropic responses were evoked by PACAP27 in guinea pig heart, depending on dose and prior exposure to the peptide. PACAP27 also caused arrhythmias in several guinea pig hearts. Treatment with atropine eliminated or prevented PACAP-evoked bradycardia and arrhythmias, implicating cholinergic neurons in these responses. Positive chronotropic responses to PACAP were unaffected by beta-adrenergic receptor blockade in either species, suggesting that tachycardia resulted from a direct action on the heart. These observations support the conclusion that endogenous PACAP could have a role in regulating parasympathetic input to the heart but through different mechanisms in rats versus guinea pigs. A direct positive chronotropic influence of endogenous PACAP is unlikely since atrial muscle lacks PACAP-immunoreactive nerve fibers.