ABSTRACTInfections with the opportunistic yeastCandida glabratahave increased dramatically in recent years. Antifungal therapy of yeast infections commonly employs azoles, such as fluconazole (FLC), butC. glabratafrequently develops resistance to these inhibitors of ergosterol biosynthesis. The pyrimidine analog flucytosine (5-fluorocytosine [5FC]) is highly active versusC. glabratabut is now rarely used clinically due to similar concerns over resistance and, a related concern, the toxicity associated with high doses used to counter resistance. Azole-5FC combination therapy would potentially address these concerns; however, previous studies suggest that 5FC may antagonize azole activity versusC. glabrata. Here, we report that 5FC at subinhibitory concentrations antagonized the activity of FLC 4- to 16-fold versus 8 of 8C. glabrataisolates tested. 5FC antagonized the activity of other azoles similarly but had only indifferent effects in combination with unrelated antifungals. Since azole resistance inC. glabrataresults from transcription factor Pdr1-dependent upregulation of the multidrug transporter geneCDR1, we reasoned that 5FC antagonism might be similarly mediated. Indeed, 5FC-FLC antagonism was abrogated inpdr1Δ andcdr1Δ strains. In further support of this hypothesis, 5FC exposure inducedCDR1expression 6-fold, and this upregulation was Pdr1 dependent. In contrast to azoles, 5FC is not a Cdr1 substrate and so its activation of Pdr1 was unexpected. We observed, however, that 5FC exposure readily induced petite mutants, which exhibit Pdr1-dependentCDR1upregulation. Thus, mitochondrial dysfunction resulting in Pdr1 activation is the likely basis for 5FC antagonism of azole activity versusC. glabrata.