A modest diet-induced increase in serum cholesterol in rabbits increases the sensitivity of the sarcolemmal Na⁺-K⁺ pump to intracellular Na⁺ while a large increase in cholesterol levels decreases the sensitivity to Na⁺. To examine the mechanisms we isolated cardiac myocytes from controls and from rabbits with diet-induced increases in serum cholesterol. The myocytes were voltage clamped using patch pipettes that contained osmotically balanced solutions with Na⁺ in a concentration of 10 mM and K⁺ in concentrations ([K⁺]_pip) ranging from 0 to 140 mM. There was no effect of dietary cholesterol on electrogenic Na⁺-K⁺ current (I_p) when pipette solutions were K⁺-free. A modest increase in serum cholesterol caused a [K⁺]_pip-dependent increase in I_p while a large increase caused a [K⁺]_pip-dependent decrease in I_p. Modelling suggested pump stimulation with a modest increase in serum cholesterol can be explained by a decrease in the microscopic association constant K_K describing the backward reaction E₁ + 2K⁺ E₂(K⁺)₂ while pump inhibition with a large increase in serum cholesterol can be explained an increase in K_K. Since hypercholesterolemia up-regulates angiotensin II receptors and since angiotensin II regulates the Na⁺-K⁺ pump in cardiac myocytes in a [K⁺]_pip-dependent manner we blocked angiotensin synthesis or angiotensin II receptors in vivo in cholesterol-fed rabbits. This abolished cholesterol-induced pump inhibition. Since the epsilon isoform of protein kinase C ([[epsilon]PKC) mediates effects of angiotensin II on the pump we included specific [[epsilon]PKC blocking peptide in patch pipette filling solutions. The peptide reversed cholesterol-induced pump inhibition.