The importance of the P1 reactive site for the specificity of ecotin on target proteases was examined by site-directed mutagenesis. The replacement of Met at the P1 site with Ile, Arg, Glu, or Tyr showed little or no effect on the ability of ecotin to inhibit trypsin. Similar results were obtained for chymotrypsin, except that its replacement with Glu caused about 40% reduction of the inhibitory activity of ecotin. On the other hand, the replacement of the Met residue with Arg, Tyr, or Glu dramatically reduced its ability to inhibit elastase, while that with Ile showed little or no effect. Nevertheless, elastase could be completely inhibited upon incubation with excess amounts of the mutant ecotin containing Arg, Glu, or Tyr. Moreover, all the mutant forms of ecotin could be cleaved at the mutated P1 site upon incubation with trypsin at pH 3.75. In addition, the replacement of a Cys residue in the disulfide bridge with Ser showed little or no effect on the ability of ecotin to inhibit trypsin, chymotrypsin, or elastase. However, the mutant ecotin containing Ser was more sensitive to inactivation by heating at 100 degrees C than the wild-type inhibitor. Furthermore, the wild-type ecotin whose disulfide bond had been reduced and alkylated was also more easily inactivated by heat treatment than the untreated control. These results strongly suggest that the P1 site of ecotin is not crucial for its specificity on target proteases and that the disulfide bridge in ecotin appears to play an important role in maintenance of its structural stability.