By indirect immunofluorescence and microcytotoxicity it was demonstrated that different populations of murine macrophages bear different amounts of Ia antigens on their membranes. At least three subpopulations could be distinguished: those that lack Ia antigens and predominate in peritoneal exudate; cells bearing I-A antigens that are the majority of splenic macrophages and a minor population in the peritoneum; and cells bearing I-C antigens that are a minor population in both spleen and peritoneum. Internal radioisotope labeling studies confirmed that the I region molecules are synthesized by the macrophages. It is suggested that these different macrophage subpopulations may play distinct roles in the immune response.