A thiazine dye reductase has been described in endothelial cells that reduces methylene blue, allowing its uptake into cells. Since a different mechanism of methylene blue uptake has been proposed in human erythrocytes, we measured methylene blue uptake and reduction in this cell type. Oxidized methylene blue (MB⁺) stimulated reduction of extracellular ferricyanide in a time- and concentration-dependent manner, reflecting extracellular reduction of the dye. Reduced methylene blue was then taken up by the cells and partially oxidized to MB⁺. Both forms were retained against a concentration gradient, and their redox cycling induced an oxidant stress in the cells. Whereas concentrations of MB⁺ less than 5 µM selectively oxidized NAD(P)H, higher concentrations also oxidized both GSH and ascorbate, especially in the absence of D-glucose. MB⁺-stimulated ferricyanide reduction was inhibited by thiol reagents with different mechanisms of action. Phenylarsine oxide, which is selective for vicinal dithiols in proteins, inhibited MB⁺-dependent ferricyanide reduction more strongly than it decreased cell GSH and pentose phosphate cycle activity, and was without effect on cellular NADPH. Open erythrocyte ghost membranes facilitated saturable NAD(P)H oxidation by MB⁺, which was abolished by pre-treating ghosts with low concentrations of trypsin and phenylarsine oxide. These results show that erythrocytes sequentially reduce and take up MB⁺, that both reduced and oxidized forms of the dye are concentrated in cells, and that the thiazine dye reductase activity initially responsible for MB⁺ reduction may correspond to an MB⁺-dependent NAD(P)H reductase activity in erythrocyte ghosts.