The role of glycolysis and oxidative phosphorylation in providing the ATP for the cardiac Na+/K+ pump was studied in cardioballs from sheep Purkinje fibres. As an indicator of the pump activity, the pump current Ip was measured at -20 mV and 30-33 degrees C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose Ip reached a maximum within 8 min and declined to 50% of this value within 27 min after gaining access to the cell interior. Perfusion with an ATP- and glucose-free medium barely enhanced the Ip decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 microM or 20 microM) moderately accelerated the effect of the ATP- and glucose-free pipette solution. Addition of 2 mM iodoacetic acid (an inhibitor of glycolysis) to the latter medium further enhanced the Ip decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of Ip to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast Ip decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 microM CCCP still hastened the fast Ip decrease slightly. This effect of complete metabolic inhibition was reversed by switching to an inhibitor-free pipette solution containing 15 mM ATP. It is concluded that the Na+/K+ pump of cardiac Purkinje cells is preferentially fuelled by glycolytic ATP synthesis.