This paper describes the initial immunochemical characterization of specific macrophage-arming factor (SMAF). SMAF is an antigen-specific factor that is released by (sensitized) T lymphocytes after contact with the specific antigen. It renders macrophages specifically cytotoxic. The specificity is dependent on the tumor-mouse combination. In allogeneic systems the specificity is H-2-directed, whereas in the syngeneic systems the specificity is tumor-specific. SMAF has a molecular mass of 65-85 kDa (established by gel filtration). By affinity chromatography SMAF could not be adsorbed with anti-(kappa + lambda light chain) immunoglobulins or anti-IgG from SMAF-containing supernatants. SMAF could be adsorbed with the monoclonal antibody 14-30 (directed against specific T-cell factors), and could be eluted from columns containing the latter. Furthermore, SMAF could also be adsorbed with and eluted from affinity chromatography columns to which specific tumor cell membranes or KCl extracts of these tumor cell membranes were coupled. Other tumor cell membranes could not adsorb SMAF. Together these data show that SMAF is not an antibody but a T-cell factor with an antigen-specific recognition site.