Abstract The efficiency of reactions involving C4 was markedly affected by antibody class, antibody concentration, temperature, ionic strength, SAC1 multiplicity, and SA:SAC1 ratio; C1-binding affinity was an important factor. Invariably, IgG-sensitized cells showed more lysis with a given amount of C4 than cells treated with IgM when other conditions were equal. Optimal reaction times and temperatures for C4 were as follows: a) for IgG-sensitized cells, 1 hr at 0°C if there are 1000 SAC1/cell, 2 hr or more at 0°C for 100 or fewer SAC1/cell; b) for high concentrations of IgM, the same conditions apply as for IgG; for lower concentrations (100 IgM/cell or less, same amount of C1), optimal conditions will vary depending on other factors (1). The above are for limiting amounts of C4. For higher concentrations of C4, times can be shortened somewhat, but the temperatures should not be altered. A further important consideration is that with IgM Ab, a 30 to 40 min preincubation of the EAC1 at 0°C before adding the C4 at 0°C markedly improves EAC14 reactivity in most cases (1). The optimal ionic strength for the above reactions is about 0.035 to 0.04. With IgM antibody, very firmly bound C1 seemed to have a reduced effectiveness in the C4 reaction, as shown by lower lysis for heavily sensitized cells, and by reduced lysis at high vs low SA:SAC1 ratios, at 30°C. This was never the case with IgG. C4 markedly increased the affinity with which C1 was bound to IgM-sensitized cells; half or more of the C1 could be bound so firmly that it failed to transfer at µ = 0.15. This effect was not seen with IgG antibody at the concentrations used, probably because smaller amounts of C4 were involved. C1-binding affinity did not appear to affect the absolute or relative hemolytic efficiencies of rat or guinea pig EDTA-C with either class of antibody.