Induction of nephrosis in rats with aminonucleoside of puromycin (ANP) was followed by an increase in urinary protease activity, measured by the cleavage of 14C-globin, as well as in antiprotease activity measured by trypsin inhibition. The excretion of protease and protease inhibitor coincided with but did not precede the onset of proteinuria when the ANP was injected subcutaneously for 5 days and lagged after the proteinuria when the ANP was given as a single intravenous dose. Serum protease activity did not change throughout ANP treatment or later, whereas serum antiprotease capacity declined coincidently with proteinuria, most probably due to the loss in urine. Kidney proteolytic activity was markedly reduced in ANP nephrosis. Treatment of rats with proteolysis inhibitors, trasylol, ε-aminocaproic acid, soybean trypsin inhibitor, or hexapron, together with ANP failed to prevent, delay or reduce the proteinuria. We believe that the urinary protease in ANP nephrosis does not originate from the circulation but from the release of kidney protease as a consequence of the glomerular lesion, and does not appear to be involved in its causation.