The effect of time of administration on excretion of two brush border enzymes--alanine aminopeptidase (AAP) and gamma-glutamyl transferase (gamma GT), and a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG) with a single high dose of vancomycin, gentamicin or a combination of vancomycin and gentamicin was studied in male Wistar rats and compared with elimination of a control group. The rats received vancomycin intraperitoneally (200 mg.kg-1), gentamicin intramuscularly (100 mg.kg-1) or the combination of the drugs by the same route. A control group received isotonic NaCl solution. The four groups of animals received a single injection at 8 a.m., 2 p.m., 8 p.m., and 2 a.m. and urine excretion values for AAP, gamma GT and NAG were determined 24 hr later. The results show that the nephrotoxicity of gentamicin + vancomycin is greater than that observed with gentamicin, which again is greater than that observed with vancomycin. Furthermore, circadian variations in renal toxicity were observed, the least occurring at 8 a.m.