Sorivudine, 1-beta-D-arabinofuranosyl-5-(E)-(2-bromovinyl)uracil, is a potent antiviral agent against varicella-zoster virus and herpes simplex virus type 1. However, sorivudine should not be used in combination with anticancer drugs such as 5-fluorouracil (5-FU) because (E)-5-(2-bromovinyl)uracil (BVU), a metabolite of sorivudine, inhibits the degradation of 5-FU, resulting in its accumulation in the blood and marked enhancement of the toxicity of 5-FU. Since phosphorolytic enzymes generate BVU from sorivudine, we investigated the distribution of the enzyme activity in rats. High activity was found in the cecal and large intestinal contents, while very low or no detectable activity in the liver, kidney, stomach, cecum, large intestine, and the stomach and small intestinal contents. These results suggest that intestinal microflora play an important role in BVU production. Therefore, we measured the phosphorylase activity in cell-free extracts from 23 aerobes, 16 anaerobes and a fungus. Bacteroides species B. vulgatus, B. thetaiotaomicron, B. fragilis, B. uniformis and B. eggerthii, dominant members of intestinal microflora, had high activity to convert sorivudine to BVU. To elucidate the contribution of intestinal microflora to BVU production in vivo, we administered sorivudine to rats treated with several antibiotics and measured the BVU concentration in the serum of rats. When sorivudine was given to rats treated with ampicillin or a mixture of bacitracin, neomycin and streptomycin, which decreased the numbers of viable aerobes and anaerobes, only a small amount of BVU was found in the serum. BVU concentration in the serum of rats treated with metronidazole to decrease the number of intestinal anaerobes was also very low. In contrast, BVU concentration in the serum of rats treated with kanamycin, which was used to decrease the number of aerobes selectively, was higher than that of non-treated rats. These results also suggest that BVU is produced by intestinal anaerobic bacteria especially Bacteroides species in vivo.