Babesia gibsoni multiplies well in canine red blood cells (RBCs) containing high concentrations of potassium (HK), reduced glutathione, and free amino acids as a result of an inherited high Na,K-ATPase activity, i.e., HK RBCs. To determine the role of Na,K-ATPase in the multiplication of B. gibsoni, the effect of ouabain on the proliferation of the parasites in HK RBCs was investigated. To determine the direct effect of ouabain on the parasites, the proliferation of the parasites in normal canine RBCs containing low potassium (LK) and high sodium concentrations, i.e., LK RBCs, which completely lack Na,K-ATPase activity, was observed. Ouabain at 0.1 mM significantly suppressed the multiplication of B. gibsoni in HK RBCs in vitro, whereas it had no effect on the parasites in LK RBCs. The results suggest that the multiplication of B. gibsoni in HK RBCs depends mainly on the presence of Na,K-ATPase in the cells. Therefore, the effects of ouabain on the intracellular cation and free amino acid composition of the HK RBCs were examined. In HK RBCs incubated with ouabain, a marked decrease in the concentration of potassium and an increase in sodium were observed, together with a decrease in the number of parasitized cells. These results suggest that the intracellular cation composition maintained by Na,K-ATPase might be advantageous to the parasites. Moreover, the concentrations of some free amino acids, i.e., asparagine, aspartate, glutamate, glutamine, glycine, and histidine, were markedly decreased in HK RBCs incubated with ouabain. Decreased concentrations of the free amino acids induced by inhibition of Na,K-ATPase seemed to affect the multiplication of B. gibsoni in HK RBCs. Based on these results, it is clear that the high Na,K-ATPase activity in HK RBCs contributes to the proliferation of B. gibsoni by maintaining high potassium and low sodium concentrations, as well as high concentrations of some free amino acids in the cells.