Human erythrocytes express Ca2+-permeable nonselective cation channels which contribute to the background leak of Ca2+ and monovalent cations. Excessive activation of these channels upon osmotic or oxidative stress initiates the death program of human erythrocytes. To define the molecular identity of these channels human erythrocyte ghosts were prepared by hemolysis in EGTA-buffered solution containing the Ca2+-sensitive dye Fluo3 and cation channel-specific antibodies and/or immunizing peptides. Ca2+ entry into the ghosts, as monitored in Ca2+-containing solution by increase of Fluo3 fluorescence, was inhibited by the reducing agent dithiothreitol (1 mM) and the erythrocyte cation channel blocker ethylisopropylamiloride (30 mM) indicating preserved cation channel function. Antibodies against TRPC6 but neither antibodies against TRPM2 nor antibodies pre-adsorbed with the immunizing peptides inhibited ghost Ca2+ entry. Accordingly, erythrocytes from TRPC6-/- mice exhibited a decreased steady state Ca2+ activity, a decreased stress-stimulated Ca2+ entry, and a decreased Ca2+-mediated phospholipid scrambling as compared to erythrocytes from TRPC6+/+ mice. In conclusion, human and mouse erythrocytes functionally express TRPC6 nonselective cation channels which contribute to both, the constitutive cation leak and the stress-triggered death program of human erythrocytes.