We have investigated the actions of vasoactive intestinal peptide (VIP) and neuropeptide Y (NPY) on the pacemaker current (If) in canine Purkinje fibers. On voltage pulses to the middle of the If activation range, VIP reversibly increases If, whereas NPY reversibly decreases If. A three-pulse voltage protocol suggests that VIP shifts If activation in the positive direction and that NPY shifts If activation in the negative direction on the voltage axis without changing maximal If conductance. These effects of VIP and NPY on If are exerted through their specific peptide receptors, since the effects are blocked by VIP and NPY receptor antagonists. VIP and NPY are colocalized in cardiac parasympathetic and sympathetic nerve endings, respectively, and can be released preferentially on high and long-lasting nerve stimulation. Given this colocalization and frequency-dependent release, these results suggest a role for these neuropeptides in controlling cardiac If and consequently heart rate.