A urinary trypsin inhibitor with an estimated molecular weight of 67,000 (UTI-1) was isolated from normal human urine by ammonium sulfate precipitation and gel filtration on Sephadex G-100. When urine was acidified prior to ammonium sulfate precipitation, two new inhibitors (UTI-11 and UTI-111) with molecular weights of 45,000 and 23,000 were obtained. The modification of UTI-1 seemed to be due to a heat-labile enzyme in native urine, since it could be prevented by heating the urine at 100 °C for 15 min. The heat labile enzyme might be a protease which was active under acidic condition, such as uropepsin.