Male Wistar rats were used to evaluate the influence of time of administration of a single high dose of vancomycin (V), gentamicin (G) or vancomycin-gentamicin combination (V/G) on excretion of a brush border enzyme, alanine aminopeptidase (AAP) and of a lysosomal enzyme, N-acetyl-beta-D-glucosaminidase (NAG). Increased urinary excretion is considered as an early manifestation of renal toxicity. The rats were placed in temperature-controlled quarters with intermittent lighting (12 hours light/12 hours dark). V was given intraperitoneally in a dose of 200 mg/kg, G was given intramuscularly in a dose of 100 mg/kg, and the V/G combination was given in the same doses and by the same routes as each drug alone. A control batch of rats received an intraperitoneal injection of saline. In the four batches, the injection was given at 8 am, 2 pm, 8 pm and 2 am. Substantial brush border toxicity was found. Toxicity of G was greatest at 2 pm and lowest at 2 am, whereas for V, toxicity was greatest at 2 am and lowest at 2 pm. With the V/G combination, brush border toxicity was greatest at 2 am and lowest at 8 am. Lysosomal toxicity was no significant after administration of V or G. In contrast, the V/G combination induced very significant lysosomal toxicity which was greatest at 2 pm and smallest at 8 am. These results show that circadian variations in renal toxicity occur not only with V and G alone but also with the V/G combination.