We studied pacemaker current (i _f), the inward current activated by hyperpolarization in rabbit sinoatrial (SA) node myocytes, with the permeabilized-patch-clamp technique. The tyrosine kinase inhibitors genistein (50 μM) or herbimycin A (35 μM) reduced the amplitude of i _f in response to step hyperpolarizations in the diastolic range of potentials. A two-step voltage-clamp protocol revealed that the reduction in i _f is due to a decrease in maximal i _f conductance. The observed effects are due to tyrosine kinase inhibition since an inactive analog of genistein did not reduce i _f. To further examine the mechanism of action, we added 2 mM chlorophenylthio cAMP (CPTcAMP, a membrane-permeant cAMP analog) to the bathing Tyrode, which increased i _f. Genistein still reduced i _f in the presence of CPTcAMP. This suggests that the pathway mediating the actions of tyrosine kinase inhibition on i _f is independent of cAMP- or protein-kinase-A-mediated phosphorylation.