The anion exchanger AE1 is the most abundant protein at the red blood cell (RBC) surface. Genetic defects of AE1 may lead to spherocytic red cell morphology and severe hemolytic anemia. The present experiments explored whether lack of AE1 triggers eryptosis, a suicidal RBC death characterized by Ca2+ entry and phosphatidylserine exposure. To this end, FACS analysis has been performed in RBC from gene targeted mice lacking AE1 (AE1(/() and from their wild type littermates (AE1+/+). Peripheral blood RBC number and packed cell volume were significantly smaller in AE1(/( than in AE1+/+ mice despite increased percentage of reticulocytes. Fluo3 fluorescence revealed markedly enhanced cytosolic Ca2+ activity, binding of fluorescent annexin disclosed enhanced phosphatidylserine exposure in AE1(/( RBC. Activation of the Ca2+-permeable cation channels by osmotic shock, Cl- removal or energy depletion increased annexin binding, effects significantly stronger in (/( RBC than in AE1+/+ RBC. The increase of annexin binding following exposure to the Ca2+ ionophore ionomycin (1 mM) was similar in AE1(/( and in AE1+/+ RBC. Thus, lack of AE1 enhances Ca2+-mediated programmed cell death, contributing to the shortened life span of the affected RBC.