In rats, renal enzyme excretion (lactic dehydrogenase = LDH; alkaline phosphatase = AP; “leucine aminopeptidase” = LAP) was determined following administration of high doses (33% of LD 50) of penicillin, kanamycin and dimethylsulfanilamidoiso-oxazole. Following injections of penicillin, only weak increases in LDH- and LAP-activities of urine occurred. As penicillin is excreted via renal tubular secretion, these observations might be the result of increased secretory activity in renal tubuli. Kanamycin and the sulfonamide lead to high increases in urinary enzyme activities, as they were encountered following administration of nephrotoxic agents. Experiments performed in vitro revealed inhibition of urinary LDH by penicillin and inhibition of urinary AP-activity by the sulfonamide. The results obtained in these series were discussed with a view to their significance in testing nephrotoxic agents by determining renal enzyme excretion. Urines of patients receiving therapeutic agents must be investigated with proper care, as drugs might activate or inhibit urinary enzymes.