The intralysosomal proteinases, cathepsins B and L, were measured in microdissected segments of rat nephrons following a single injection of puromycin aminonucleoside (PAN). Z-Phenylalanyl-arginine-7-amido-4-methylcoumarin served as substrate. Enzyme activities, proteinuria, creatinine clearance and renal morphology were determined at specific time intervals following induction of PAN nephrosis. During the first three days following PAN injection, enzyme activities in S2 and S3 segments, protein excretion, creatinine clearance and appearance of the renal parenchyma resembled control animals. The enzyme activity in S1 segments was slightly decreased, but returned to control levels at day six after injection. Days four through eight post-PAN injection were characterized by a dramatic increase in protein excretion and an increase in cathepsin B and L activity in S2 and S3 segments of the proximal tubule. During days 9 through 15 enzyme activity decreased significantly in S2 segments despite continued proteinuria. Overt necrosis and cell injury were seen in the proximal tubule and probably account for the decrease in proteolytic activity. After day 15 following PAN injection, the level of proteinuria decreased, restoration of cathepsin activities occurred and a histopathologic picture of healing was present. The data suggest a positive relationship exists between stimulation of cathepsin B and L activity in S2 and S3 segments of the proximal tubule and increased protein filtration in PAN nephrosis. The increased enzyme activity reflects enhancement of the proteolytic capacity of the lysosomal system that is necessary for increased protein catabolism.