Isoproterenol reversibly depolarizes quiescent sheep cardiac Purkinje fibers, in contrast to its reported hyperpolarizing effect in many excitable tissues. The depolarization is inhibited by drugs that block beta 1-adrenergic receptors. Tetrodotoxin and verapamil have no effect on the isoproterenol-induced depolarization. Cesium reduced the isoproterenol-induced depolarization by 74%. The voltage dependency of activation of a current component called If, measured under voltage clamp, was shifted in the depolarizing direction by isoproterenol. No evidence was found to support the suggestion made for other tissues that the Na+-K+ pump is stimulated by isoproterenol. These data suggest that in quiescent sheep Purkinje fibers the isoproterenol-induced depolarization reflects activation of a pacemaker current.