Abstract A trypsin inhibitor (UTI) was isolated from pooled urine from pregnant women by DEAE (diethylaminoethyl)-cellulose chromatography with a yield of 30 per cent and purified to a nearly homogeneous preparation. The majority of the antitrypsin activity in urine appears to be due to a single inhibitor which is a glycoprotein containing approximately 12 per cent carbohydrate with an estimated molecular weight of 70,000. The original crude fractions contained a heat-labile enzyme which under acid conditions rapidly modified UTI to a less anionic form without affecting the trypsin inhibitor activity. The inhibitor isolated in the present study has a significantly larger molecular weight than inhibitors prepared by other investigators. The procedures used by previous workers included an initial exposure of the crude inhibitor to acid conditions. Our results suggest that the UTI obtained in earlier studies probably represented the isolation of the modified inhibitor(s). From the results of this investigation, it is not yet possible to interrelate UTI to any of the known plasma trypsin inhibitors. The origin of UTI in urine remains a project of future research.