Abstract A cell surface antigen (AMøCSA) associated with activated macrophages was induced in vivo on murine peritoneal macrophages by pyran copolymer or Corynebacterium parvum but not by glycogen or thioglycollate. The antigen was detected by complement-dependent cytotoxicity by using an appropriately absorbed rabbit antiserum to the P388D1 macrophage cell line. Antigen was detectable on 90 to 100% of peritoneal macrophages 6 hr after i.p. inoculation of pyran or C. parvum and gradually declined to background levels, both with respect to the number of positive cells and the amount of antigen per cell, by 30 days after induction. Activated macrophages treated with pronase but not trypsin, neuraminidase, or β-galactosidase were no longer susceptible to lysis with AMøCSA-specific antiserum and C, but they could still be lysed with an antiserum directed to an organ-specific macrophage antigen shared by both normal and activated macrophages. Studies with four bacterial vaccines, Propionibacterium acnes, type I; two strains of P. acnes, type II, and P. granulosum have indicated that only two vaccines, P. acnes, type I and one strain of P. acnes, type II, could induce: a) regression of the MCA 2182 fibrosarcoma when inoculated intralesionally, b) splenomegaly, and c) peritoneal macrophages cytotoxic to tumor cells in vitro. The new antigen associated with activated macrophages was only detectable on macrophages activated by the two vaccines, P. acnes, types I and II, that induced functionally activated cells. These results are consistent with the induction of a new cell-surface antigen associated with activated but not with normal or elicited macrophages.