Abstract: The antifungal flucytosine (5-fluorocytosine; 5FC) is a prodrug metabolized to its toxic form 5-fluorouracil (5FU) only by organisms expressing cytosine deaminase. One such organism is Candida glabrata, which has emerged as the second most common agent of bloodstream and mucosal candidiasis. This emergence has been attributed to the high rate at which C. glabrata develops resistance to azole antifungals. As an oral agent, 5FC represents an attractive alternative or complement to azoles; however, the frequency of 5FC-resistance mutation and mechanisms by which these mutations confer resistance have been minimally explored. On RPMI medium containing 1 mug/ml 5FC (32-fold above the MIC, but less than one-tenth of typical serum levels), resistant mutants occurred at relatively low frequency (2 x 10(-7)). Three of six mutants characterized were 5FU cross-resistant, suggesting a mutation downstream of Fcy1 (cytosine deaminase), which was confirmed by sequence analysis of Fur1 (uracil phosphoribosyl transferase). The remaining 3 had Fcy1 mutations. To ascertain the effects of 5FC-resistance mutations on enzyme function, mutants were isolated in ura3 strains. Three of seven mutants harbored Fcy1 mutations and failed to grow in uridine-free, cytosine-supplemented medium, consistent with inactive Fcy1. The remainder grew in this medium and had wild-type Fcy1; further analysis revealed these to be mutated in the Fcy2L homolog of S. cerevisiae Fcy2 (purine-cytosine transporter). Based on this analysis, we characterized three 5FC-resistant clinical isolates: mutations were identified in Fur1 and Fcy1. These data provide a framework for understanding 5FC resistance in C. glabrata and potentially other fungal pathogens.