Macrophages (M phi) were recovered from disaggregated, spontaneously regressing Moloney sarcomas for in vitro testing of their cytotoxic capabilities. At a 3:1 ratio, M phi in monolayers efficiently killed (51Cr release) a variety of tumor target cells without regard to antigenic specificity. Within 24 hr most of their cytolytic potential was lost. Killing could be restored, however, if M phi were replated at higher cell densities. A soluble, heat stable, dialyzable inhibitor of thymidine incorporation, which did not interfere with cellular proliferation, also was produced by M phi recovered from Moloney sarcomas. The finding of such a competitive inhibitor, probably cold thymidine, in supernatants further opened to question the validity of radioisotope incorporation into DNA as a measure of M phi mediated cytostasis. The data presented suggest that M phi may participate in the regression process by directly killing tumor cells, thus complementing the antigen specific cytolytic capabilities of the T lymphocytes which are also found in large numbers in regressing Moloney sarcomas.