The hypothesis that sublethal doses of aminoglycosides cause renal tubule disorders resulting in changes of urine enzyme levels was investigated. The renal status following injection of a single sublethal dose of gentamicin (200 mg/kg) at different times during a 24 h cycle was studied. Increased excretion of gamma-glutamyl transferase, alanine aminopeptidase and N-acetyl-beta-D-glucosaminidase, used clinically as markers for tubule toxicity of aminoglycosides, was maximal when gentamicin was administered to rats at 2 p.m. and was minimal when injected at 8 p.m. These significant differences in enzyme excretion as a function of injection time are correlated with the concentration of gentamicin in the urine and in the renal cortex.