A trypsin inhibitor was purified 4,100-fold to apparent homogeneity from Drosophila melanogaster. The molecular weight of the purified protein inhibitor was estimated to be 8,000 daltons by gel permeation chromatography, and 8,500 daltons by sodium dodesyl sulfate-polyacrylamide gel electrophoresis. The inhibitor forms a complex with bovine pancreatic trypsin at a molar ratio of 1:1. This protein can inhibit other proteases, such as porcine pancreatic elastase and collagenase, but not chymotrypsin, subtilisin, papain, or pepsin. The inhibitor retained its activity over a broad range of pH (1 to 12) and was relatively thermostable.