AbstractThe time and temperature requirements of certain steps involved in hemolysis of normal human red cells by cold‐reacting antibodies and normal human serum were examined by means of techniques that measure the amount of antibody and the fourth (C4), third (C3), and fifth (C5) component of complement bound to red cell membranes. For all cold‐reacting antibodies, the greatest degree of lysis was obtained in bithermic reactions with the first phase at temperatures 15° or below, and the second phase at 37°. For anti‐I antibodies, only antibody and C1 are fixed at temperatures below 15°, and the fixation of C4 and C3 to the membrane occurs during warming. C4 but not C3 is fixed at temperatures below 15° when the reaction is initiated by the Donath‐Landsteiner antibody. During warming, antibody and C1 elute. No C4 and C3 are fixed after the temperature reaches 37° and the amount of “active” C3 diminishes rapidly with continued incubation at 37°. C5 is rapidly fixed during warming from 15° to 37° and for a short time at 37°, but the hemolytic activity of C5 is unstable. Thus these cold‐reactive antibody‐mediated complement reactions occurring on human red cells in human serum must occur during and very shortly after the warming phase. If the rate of change of temperature is prolonged during the warming phase, greater C3 fixation and hence more lysis occurs.