Evidence is presented that interferon (IF) is a major mediator of the human concanavalin A (Con A) suppressor cell. The suppressive effects of Con A-activated lymphocytes on the mitogen responses of normal responder cells were largely abrogated by addition of anti-human leukocyte IF serum. Similar suppressor activity was generated by coculture of peripheral blood leukocytes (PBL) with a melanoma cell line (MeWo) and a HeLa cell line persistently infected with measles virus that induced the production of IF by lymphocytes. A human mammary carcinoma line (MCF-7) and two bladder carcinoma lines (T24 and TCCSUP) failed to induce IF or suppression. Addition of anti-human leukocyte IF serum to suppressor cells and supernates from tumor cell-lymphocyte cocultures largely abolished suppression and neutralized the antiviral activity of such supernates. Exposure of PBL from purified protein derivative (PPD)-positive donors to PPD caused the production of suppressor activity and IF. PBL from PPD-negative donors failed to produce significant amounts of IF or to suppress on exposure to PPD. Supernates from PBL treated with virus (Newcastle disease virus [NDV]) contained IF and suppressed the mitogen responses of responder PBL. Both the suppressive and the antiviral activities of this material were eliminated after treatment with anti-IF serum. To ascertain whether antiviral and suppressive activities were mediated by the same types of IF, supernates from PBL cultured with Con A, PPD, NDV, and tumor cells were treated with anti-IF serum or acid pH. In all cases antiviral activity was neutralized in parallel with abrogation of suppressor activity. These results provide strong evidence for the role of IF as a mediator of human suppressor cell activity.