Purified human C3 was found to inhibit rat in vitro secondary antibody responses. Fifty percent inhibition of antibody-forming cell development occurred with C3 concentrations of 26 micrograms/ml. This decrease was not the result of a general toxicity or a shift in the antibody response kinetics. Using cell mixing experiments, we could not detect a C3-induced suppressor lymphocyte or macrophage. C3 was active when added to culture early (day 0 or 1 or during a 24-hr antigen prepulse) or late (day 3, 5, or 7)--the early addition being more suppressive. Regardless of the addition time, there was a characteristic 48- to 72-hr lag before the inhibitory effect was manifested. C3 could inhibit antibody-forming cell development after stimulation with the thymus-independent antigens, trinitrophenyl-Brucella abortus and dinitrophenyl-Ficoll, as well as the thymus-dependent antigens, dinitrophenyl-bovine gamma-globulin and chicken gamma-globulin suggesting that C3 was not selective for B memory cell subpopulations. Further characterization of our C3 preparation indicated that the majority of the suppressive activity resided in a small m.w. protein resembling the C3a fragment of C3. Human C3a preparations generated either by trypsin cleavage or zymosan activation of C3 were also tested in our antibody response system and were able to inhibit antibody-forming cell development. These data implicate C3 cleavage products as negative regulators of antibody formation.