The mechanism of synergistic complement-mediated lysis of rat red cells was investigated using rat monoclonal antibodies against class I RT1Aa antigens. The increased lytic activity when using two antibodies simultaneously is due to the increase in the number of activated C1 molecules on the cell surface and this results from (a) an increase in the number of binding sites for C1q, (b) an increase in the functional affinity constant for C1q binding and (c) an increase in the rate of activation of C1. Complete lysis of red cells was only achieved if one member of the synergistic pair was of the gamma 2b isotype, and this isotype was the only one to which binding of 125I-labeled C1q could be detected. A partial synergistic effect was seen using an F(ab')2 fragment of antibody. Increased uptake and activation of C1 probably results both from the presence of two antibodies attached to each antigen molecule and from the formation of antigen-antibody catenars.