Human alpha-thrombin, the procoagulant activation product of prothrombin, elicits chemotaxis in human peripheral blood monocytes and several macrophagelike continuous cell lines, most notably J-774.2, but not in human peripheral blood granulocytes. alpha-Thrombin is effective in stimulating cell movement at concentrations ranging from 10(-10) to 10(-6) M but is optimally active at 10(-8) M. At the latter concentration, the degree of response is equivalent, on a molar basis, to that observed with the peptide formylmethionylleucylphenylalanine, (FMP). In contrast to thrombin, prothrombin produces a minimal chemotactic response in monocytes and J-774.2. Blockade of alpha-thrombin's active center with diisopropylfluorophosphate (DIP-F) or tryptic proteolysis of the procoagulant exosite (i.e., gamma-thrombin) fails to alter chemotactic activity. On the other hand, addition of equimolar amounts of antithrombin III (AT3) to alpha-thrombin reduces thrombin-mediated chemotaxis by 60%, and increased ratios of AT3 to enzyme completely suppress chemotaxis. We conclude that thrombin is a potent monocyte chemotaxin and that the domains in thrombin involved in stimulating cell movement are distinct from the catalytic site and the fibrin recognition exosite. These chemotactic domains appear to be sequestered in prothrombin and in the thrombin-AT3 complex and, as such, are unavailable to the chemotactic receptor on the monocyte cell membrane.