Single Purkinje cells from dog, sheep and cow hearts were isolated by injecting a Ca-free collagenase containing Tyrode solution in the space between the connective tissue sheath and the Purkinje cells. A small proportion of these cells survived the isolation procedure and these cells were used for further investigation. The cells showed electrophysiological properties similar to intact Purkinje fibres as indicated by the following results. Maximum diastolic potentials between -70 and -85 mV and specific membrane resistances of 21-32 k omega cm2 indicated that the single cells were not leaky or hyperpermeable . The action potential showed a rapid upstroke, with a maximum rate of rise, Vmax' between 150 and 750 V/s, and two phases of fast repolarization separated by a plateau phase with a duration of about 200 ms. Each action potential was followed by a spontaneous depolarization with an amplitude between 1 and 10 mV. The upstroke of the action potential could be blocked by tetrodotoxin (TTX) in a dose-dependent manner. The rate of depolarization of the action potential was sensitive to changes in membrane potential; the resulting S-shaped curve showed a half-maximum potential of -65 mV and a steepness of 0.46 mV-1. The duration of the action potential was sensitive to external K concentrations, catecholamines and TTX in a way similar to intact Purkinje fibres. Both application of catecholamines and lowering the external K concentration induced spontaneous activity. The cells were used to study the ionic nature of the pace-maker current under voltage-clamp conditions using the two-micro-electrode technique. This pace-maker current was blocked in a voltage-dependent manner by 1 mM-Cs, and was not affected by 1 mM-Ba. The steady-state activation curve was shifted in the depolarizing direction by application of adrenaline. In contrast to voltage-clamp data obtained on the pace-maker current of intact Purkinje fibres, the pace-maker current in a single cell did not reverse near the presumed equilibrium potential for K ions; no reversal could be seen in the voltage range negative to -50 mV. These observations together with preliminary results on the Na and K dependence of the pace-maker current are strong arguments in favour of the hypothesis that the pace-maker current in cardiac Purkinje fibres is an inward current carried by Na and K ions and activates upon hyperpolarization.