In cultures of spleen cells from mice immunized with horse erythrocytes (HRBC) 7 days earlier, the simultaneous addition of sheep erythrocytes (SRBC) and the priming antigen on day 0 resulted in the suppression of the anti-SRBC plaque-forming cell (PFC) response by day 5 compared to the response of similar cultures that received only SRBC. The cell-free culture fluid from specifically-stimulated, HRBC-primed cells, but not normal cells, contained a factor that nonspecifically inhibited the anti-SRBC PFC response of cultures of normal spleen cells to which SRBC and diluted supernatant aliquots were added at the beginning of culture. Suppressor activity was not manifest unless active supernatants were added on day 0 of a 5-day culture period. Inhibition of the reference plaque response was not due to cytotoxicity of the active material, decreased immunogenicity of the SRBC, or switchover from IgM to IgG plaque formation. The soluble mediator was released slowly into the culture fluid, with linear kinetics, from specifically activated, primed cells, with maximum suppression obtained with the 120-h supernatant. When active supernatants were fractionated by gel filtration over Sephadex G-150, the inhibitory factor eluted with molecules of about 34,000 mol. wt.