The mechanism of toxicity from 5-fluorocytosine chemotherapy is unclear. However, recent evidence suggests that the generation of 5-fluorouracil by a host may play an important role in the development of this toxicity. Using an in vitro semicontinuous culture system to mimic the intestinal microflora, we examined the capacity of this complex microbial community to convert 5-fluorocytosine to 5-fluorouracil. The system was dosed initially and after 2 weeks of chronic exposure to 5-fluorocytosine with radiolabeled 5-fluorocytosine. No detectable production of 5-fluorouracil was observed up to 8 h after the acute dose; however, at 24 h and at all time points thereafter, increasing levels of 5-fluorouracil were detected for 4 days. The chronic dose resulted in an increased rate of 5-fluorouracil production without the 8-h lag time. These findings suggest that the enzyme or enzymes responsible for the deamination of 5-fluorocytosine to 5-fluorouracil by the intestinal microflora can be induced by chronic exposure to 5-fluorocytosine and that this conversion may provide a mechanism through which 5-fluorocytosine toxicity is manifested.