The actions of the phosphatase inhibitor calyculin A on the pacemaker current i(f) were studied in canine Purkinje fibers and myocytes. Calyculin A increased i(f) in response to hyperpolarizations toward the middle of the i(f) activation curve. A three pulse protocol indicated this increase was due to a positive shift of i(f) activation on the voltage axis. Taken together with our previous results (that kinase inhibition with H7 shifts i(f) activation in the negative direction on the voltage axis (2)), these results suggest that phosphorylation is an important regulator of the voltage dependence of i(f) activation.