Summary: Over a wide range of ionic strengths the first component of complement (C′1a) was found to be readily eluted from IgG-sensitized cells at temperatures above 0°C. Even at 0°, loss of C′1a was high at ionic strengths appreciably above 0.037. With IgM Ab the effects of ionic strength were similar, but those of temperature were reversed: C′1a was increasingly better retained on cells as the temperature increased from 0° to 37°C. Conditions for reaching optimal C′1a activity with both classes of antibody (Ab) were determined. The optimal ionic strength for transfer of C′1a from EAC′1a to EAC′4 (red cells sensitized by Ab and the first and fourth components of complement, respectively) was found to be approximately 0.115 for both IgG and IgM Ab. When all conditions of C′1a fixation, washing and transfer were optimal, essentially 100% of the C′1a originally bound to sensitized cells could be accounted for by transfer to EAC′4, regardless of the class of Ab used. Transfer of C1a from IgG-sensitized cells was appreciably better at 30°C than at 0°, but with IgM Ab the converse was true. Kinetic studies showed that transfer was essentially complete within 5 to 15 min. C′1a dissociated less readily from EAC′1a, 4 than from EAC′1a, indicating that cell-bound C′4 contributes to the bond between C′1a and its site of attachment. In contrast to the situation with IgM Abs, with IgG it was not possible to reach a saturation level of C′1a; with each increase in amount of C′1a added, there was an increase in the number of SAC′1a generated, even when the ratio of C′1a:SAC′1a exceeded 100:1.