On exposure to endotoxin and other stimuli, human peripheral-blood mononuclear cells generate a potent procoagulant activity (PCA), identified as tissue factor. Although it is now recognized that the monocytes are the source of PCA, the question whether these cells per se are capable of procoagulant response to endotoxin or require lymphocyte collaboration remains unsettled. We have investigated the capacity of highly purified human macrophages from diverse anatomical sites to generate PCA following endotoxin stimulation. Purified (greater than 99%) monocyte-derived macrophages were obtained by prolonged (3-10 days) in-vitro culture of adherent monocytes using medium supplemented with 50% human serum. Purified (greater than 95%) peritoneal and milk macrophages were isolated by adherence to plastic. PCA was measured before and after incubation (4 hr at 37 degrees) with endotoxin (Salmonella enteritidis LPS, W or Escherichia coli O111: B4LPS, W, 1 microgram/ml final concentration) using a one-stage clotting assay and/or a two-stage amidolytic assay. Monocyte-derived macrophages had low baseline PCA (14-19 units/10 (5) cells) but, upon exposure to endotoxin, displayed an eight-fold increase in PCA over control. Peritoneal and milk macrophages expressed very low baseline activity (1-5 units/10 (5) cells). The latter, however, increased 15-20 times over control following endotoxin stimulation. PCA was identified as tissue factor by biological and immunological criteria. Its generation was completely abolished by cycloheximide. It is concluded that in the human mononuclear phagocyte series the capacity to produce PCA is not restricted to circulating monocytes but is also expressed by macrophages obtained from diverse anatomical sites. These macrophages appear to be autonomous in their procoagulant response to endotoxin.