Two monoclonal antibodies, M3/31 and M3/38, were obtained by fusion of mouse myeloma cells with rat spleen cells immunized to immunoadsorbent-purified macrophage glycoproteins. Co-precipitation experiments show that antigenic determinants recognized by these two antibodies reside on the same molecular species, termed Mac-2, Mac-2, an antigen of 32,000 Mr, is synthesized by and expressed on the surface of thioglycollate-elicited macrophages as shown by [35S]-methionine and 125I labeling. Saturation binding experiments show that thioglycollate-elicited macrophages express 1.7 X 10(5) Mac-2 sites/cell. Thioglycollate-elicited macrophages are strongly absorptive for 125I-labeled M3/38 MAb. Kidneys are also absorptive; however, evidence is presented pointing to the nonspecificity of this absorption. Lymph node and thymus are negative, whereas spleen and bone marrow are weakly absorptive, probably due to stromal cells. Nonlymphoid tissues, such as lung, liver, heart, and brain, exhibit slight or no absorbing capacity. Cell suspensions from spleen, bone marrow, thymus, and peripheral lymph node are greater than 99% Mac-2- by immunofluorescent flow cytometry. In contrast, thioglycollate-elicited macrophages are greater than 96% strongly positive for Mac-2. Only 20% of peptone-elicited cells are weakly positive, whereas resident peritoneal macrophages and other macrophage elicited by Listeria monocytogenes, Con A, or LPS are greater than 98% negative. SDS-PAGE of [35S]-methionine-labeled Mac-2 shows that thioglycollate-elicited macrophages synthesize 10- to 30-fold more Mac-2 than other peritoneal macrophage subpopulations, whereas all types of peritoneal macrophages synthesize and express on their surfaces similar amounts of the Mac-1 antigen. Mac-2 antigen is therefore induced in macrophages only in response to specific differentiative signals.