To determine the basis for age-related changes that occur in the transmembrane action potentials (AP) of canine cardiac Purkinje fibers (PF), we used standard microelectrode techniques to study PF from normal neonatal and adult beagles. As previously shown, neonatal PF AP have a less negative maximum diastolic potential (MDP), a lower AP amplitude (amp) and maximum upstroke velocity of phase 0 (Vmax), and shorter AP duration measured at 50% and 100% repolarization than have those of adults. Further, the termination of phase 1 and the peak of phase 2 occur at more positive levels of membrane potential in neonatal PF. The less negative MDP in neonatal PF is associated with a lower intracellular potassium activity (aK′) as demonstrated with a K+-sensitive microelectrode. Neonatal PF showed greater depression of AP amp and Vmaxduring superfusion with the rapid Na+channel blocker, tetrodotoxin (TTX), than did adult PF. The attainment of more negative voltages at the termination of phase 1 in adult compared to neonatal PF was not attributable to age-related differences in response to changes in drive cycle length, temperature, or external Cl” concentration. However, 4-aminopyridine, a blocker of the early, repolarizing potassium current, altered this variable in adult and not in neonatal PF. The effects of verapamil and AHR-2666, both slow inward current blockers; of TTX, which blocks a background inward Na+current; and of TEA+, which blocks repolarizing K+current, were studied on AP repolarization. Adult PF AP were more sensitive to the effects of AHR-2666, verapamil, and TEA* and less sensitive to the effects of TTX than neonates.