The time course of the interactions of alpha 2-macroglobulin with trypsin and with plasmin was studied by measuring the generation of thiol groups, the concentration of alpha 2-macroglobulin subunits cleaved at the bait regions, and the change in intrinsic protein fluorescence of alpha 2-macroglobulin-enzyme reaction mixtures as functions of time. The interaction of alpha 2-macroglobulin with trypsin was found to be very fast but could be studied in the presence of benzamidine, a rather strong competitive inhibitor of trypsin. The results obtained indicate that alpha 2-macroglobulin-proteinase reactions, known to involve specific limited proteolysis of the bait regions, gross conformational changes, and cleavage of the internal beta-cysteinyl-gamma-glutamyl thiol esters of native alpha 2-macroglobulin, may proceed via at least two different reaction pathways determined by the nature of or the concentration of the reacting proteinase. After initial cleavage of one bait region at high proteinase activity the next step presumably is a fast cleavage of a second bait region before any substantial rearrangements leading to generation of thiol groups and the final incorporation of the proteinase occur. At low proteinase activity no further bait region cleavages occur and only the two thiol groups of half of the alpha 2-macroglobulin molecule are generated in the final 1:1 complex.(ABSTRACT TRUNCATED AT 250 WORDS)