A protease inhibitory activity has been detected in the hemolymph of the horseshoe crab, Limulus polyphemus. The inhibitor possesses many of the characteristics of vertebrate alpha 2-macroglobulin. The inhibitor, found in the high speed supernatant of Limulus plasma, is sensitive to mild acidification, inhibits a variety of prokaryotic and eukaryotic endopeptidases, and is sensitive to methylamine treatment. Although the inhibitor completely prevents the trypsin-mediated hydrolysis of protein substrates, the hydrolysis of a low molecular substrate by trypsin is unaffected by the inhibitor, a property characteristic of alpha 2-macroglobulin. The striking similarity of the inhibitor to vertebrate alpha 2-macroglobulin, coupled with the fossil record of Limulus, suggests that such protease inhibitors are ancient molecules.