The mode of interaction between α2-macroglobulin and trypsin [EC 3.4.4.4] has been investigated with the use of various types of substrates and inhibitors. When trypsin was mixed with α2-macroglobulin, a complex was immediately formed and it was deduced to be 3: 1. By binding to α-macroglobulin, hydrolytic activity of trypxsin on TAME**** was not affected but hydrolytic activity on TLME decreased to 65%. From kinetic analyses, it was found that maximum velocities (Vmax of hydrolysis of BAEE and BAPNA by trypsin were decreased to 23% and 52%, respectively, by α2-macroglobulin. α2-Macroglobulin strongly inhibited the proteolytic activity of trypsin. Caseinolytic and fibrinogenolytic activities were observed to be decreased by 91% and 100%, respectively. Esterolytic activity of the α2-macroglobulin-trypsin complex was not blocked by protein inhibitors, such as SBTI, EWTI or LBTI, but was blocked by the low molecular weight polypeptide inhibitor, Trasylol, or active site specific inhibitors, such as TLCK and DFP. These results suggested that no part of the active site region in trypsin was implicated in the binding to α2-macroglobulin. From these results, a possible mode of interaction between α-macroglobulin and trypsin was presented as follows; in the α-2-macroglobulin-trypsin complex the catalytic site lies in a crevice. Low moleculart weight synthetic substrates or inhibitors can diffuse into the crevice, whereas bulkier protein substrates or inhibitors can not. Acid treatment of the α-macroglobulin-trypsin complex at pH2.0 restores the accessibility of the catalytic site to protein substrate as well as protein inhibitors. By the acid treatment, about 40% of the total amount of trypsin was dissociated from the complex. Since it was observed that the α2-macroglobulin-trypsin complex dissociates in acidic solution and that trypsin binding activity of α2-macroglobulin was decreased by treatment with methylamine, it was supposed that carboxylate goups in α2-macroglobulin might be implicated in the binding of trypsin.