Abstract Paroxysmal nocturnal hemoglobinuria (PNH) is an acquired hemolytic anemia. The abnormal PNH erythrocytes are highly susceptible to complement-mediated lysis in vitro, especially at pH 6.4. Lysis has been shown to be due to alternative pathway activation. The purpose of this study was to determine why lysis of PNH erythrocytes is increased at acidic pH. The results presented demonstrate that at pH 6.4: binding of C5 and Factor B to C3b deposited on human erythrocytes is markedly enhanced; generation of the two C3 convertases, C3(H2O), Bb and C3b,Bb is increased; and control of C3b on human erythrocytes by CR1 and Factor I is diminished. In addition, it was found that rabbit erythrocytes, which activate the human alternative pathway, are also lysed much better at pH 6.4 than at pH 7.4. These results indicate that the optimal pH for the initiation and amplification of the alternative complement pathway, and probably also for the activation of the membrane attack complex, is 6.4.