Proteinase inhibitor isolaled from horsegram (Dolichos biflorus or Macrotyloma uniflorum) inhibited specifically the enzymes trypsin and chymotrypsin. The inhibitor contained seven disulfide linkages and was free from thiol groups. The inhibitor is resistant to denaturation by urea, guanidine hydrochloride or sodium dodecyl sulfate. Reduction of the inhibitor with dithiothreitol abolished both trypsin and chymotrypsin inhibitory activities. The kinetic plots of the reduction as followed by activity and loss in structure as reflected in the 257 nm CD band could be superposed; loss in the activity paralleled the loss in structure. The kinetics of the reduction process was complex; reduction of the inhibitor was slow and depended on the concentration of DTT. Reduction of the disulfide linkages with DTT affected the tertiary structure: significantly and secondary structure was not affected considerably. Fluorescence quenching by acrylamide and potassium iodide suggested the unfolding of the molecule due to reduction. Thus, disulfide linkages play a predominant role in maintaining the three-dimensional structure of the inhibitor.