Tryptase, the dominant protease in human mast cells, was examined for its effect on human prekallikrein. Tryptase in the presence and absence of heparin failed to activate prekallikrein as shown in a spectrophotometric assay for kallikrein employing benzoy 1-pro-phe-arg-p-nitroanilide. Treated prekallikrein was converted to active kallikrein by bovine trypsin. Prekallikrein cleavage products were analyzed by electrophoresis in polyacrylamide gels under denaturing conditions (+/- reduction). Tryptase caused no apparent cleavage under conditions where trypsin caused complete cleavage. Thus, tryptase, which has previously been shown to lack kallikrein and kininase activities, neither activates nor destroys prekallikrein.