Alterations in membrane cholesterol induced in vitro can alter Na+-K+ pump function. Because dietary cholesterol can influence membrane cholesterol in vivo, we examined if dietary cholesterol is a determinant of Na+-K+ pump function. Rabbits were fed cholesterol-supplemented diets for 1-4 wk. Cardiac myocytes were then isolated, and Na+-K+ pump currents (Ip) were measured using the whole cell patch-clamp technique. When the Na+ concentration in the patch pipettes ([Na]pip) was 10 mM, a modest diet-induced increase in serum cholesterol was associated with stimulation of Ip; large increases in serum cholesterol were associated with inhibition. There was no effect of modest or large increases in serum cholesterol on Ip when [Na]pip was 80 mM. The [Na]pip-Ip relationship determined using seven different levels of [Na]pip from 0 to 80 mM indicated that a modest increase in serum cholesterol increased the apparent affinity of the pump for cytoplasmic Na+. In contrast, dietary cholesterol had no effect on the apparent affinity of the pump for extracellular K+. We conclude that cholesterol intake influences the sarcolemmal Na+-K+ pump. This may have clinical implications for cardiovascular function.