The occurrence of DNA synthesis in mouse peritoneal macrophages was estimated autoradiographically in cells cultured on glass in the presence of tritiated thymidine. Subcutaneous injection of a soluble antigen, human serum albumin (HSA) into mice previously immunized with HSA in Freund’s complete adjuvant was followed, after a lag of at least 16 h, by the onset of DNA synthesis in peritoneal macrophages. This effect was immunologically specific and was accompanied by morphological changes indicating activation. The ability to respond in this way was passively transferable by means of spleen cells, but not serum, from immunized mice. DNA synthesis in macrophages was induced in vivo by Concanavalin A, phytohaemagglutinin-P, anti-lymphocyte globulin, bacterial endotoxin and normal mouse serum. The number of macrophages synthesizing DNA was also increased in mice with graft-versus-host reactions and in mice bearing a syngeneic methylcholanthrene-induced tumour.