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 I _p was measured at -20mV and 30–33° C by means of whole-cell recording. During intracellular perfusion with a pipette solution containing 5 mM ATP and 15 mM glucose I _p 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 I _p decline. Inhibition of the oxidative phosphorylation by carbonylcyanide m-chlorophenylhydrazone (CCCP, 2 μM or 20 μM) 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 I _p decrease with time. Inhibition of the glycolytic ATP synthesis by 2-deoxy-D-glucose (5 mM) caused a dramatic decline of I _p to half of its maximum within 7.3 min. Pyruvate (5 mM) and inorganic phosphate (2 mM) did not affect the fast I _p decline evoked by the ATP- and glucose-free, 2-deoxyglucose-containing medium, whereas 2 μM CCCP still hastened the fast I _p 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.