Cardiac pacemaker current, if, is generated by hyperpolarization-activated cyclic nucleotide-gated (HCN) channels. Our previous studies demonstrated that altered tyrosine phosphorylation can modulate the properties of both if and HCN channels. To assess a hypothesis that the intracellular tyrosine kinase Src may play a role in modulation by tyrosine phosphorylation of if, we cotransfected HEK293 cells with HCN4 and Src proteins. When HCN4 was cotransfected with a constitutively activated Src protein (Src529), the resultant voltage-dependent HCN4 activation was positively shifted (HCN4: V1/2 = -93 mV; Src529: V1/2 = -80 mV). The activation kinetics were accelerated at some potentials but not over the entire voltage range tested (eg, at -95 mV, tau_act(HCN4) = 3,243 ms; tau_act(Src529) = 1,113 ms). When HCN4 was cotransfected with a dominant negative Src protein (Src296), the HCN4 activation was shifted more negative to a smaller degree (HCN4: V1/2 = -93 mV; Src296: V1/2 = -98 mV; statistically insignificant) and the activation kinetics were slowed at most test potentials (eg, at -95 mV, tau_act(Src296) = 7,396 ms). Neither Src529 nor Src296 significantly altered HCN4 current density. Coimmunoprecipitation experiments revealed that Src forms a complex with HCN4 in HEK293 cells and in rat ventricular myocytes. Our data provide a novel mechanism of if regulation by Src tyrosine phosphorylation.