Soluble immune response suppressor (SIRS) is a product of concanavalin A activated Ly 2,3+ T cells which suppresses plaque-forming cell response to sheep erythrocytes by murine spleen cells in vitro through its action on macrophages. The mechanism(s) by which SIRS-treated macrophages suppress plaque-forming cell responses has been investigated. Experiments with varying ratios of control supernatant- and SIRS-treated macrophages showed that suppression was not due to inactivation of macrophages, but was an active process. This active process required at least 3 days for full expression since: 1) responses in cultures containing SIRS-treated macrophages developed normally for 3 days and suppression became apparent only on day 4, approximately 24 hr after a significant decrease in DNA synthesis; 2) responses of spleen cells in a Millipore chamber which were incubated with SIRS-treated macrophages for 48 hr and then transferred to control supernatant-treated macrophages for the last 72 hr of culture escaped suppression; and 3) addition of SIRS-treated macrophages to spleen cell cultures after initiation resulted in later expression of suppression. The experiments in which spleen cells were incubated in Millipore chambers separated from SIRS-treated macrophages indicated that a second factor(s), released from the SIRS-treated macrophages, was active in suppressing the response. This factor may also be responsible for the decrease in DNA synthesis after day 3 of culture and suggests that SIRS-mediated suppression is ultimately due to limitation of proliferation of responding B cells.