Oral fungal infections are caused commonly by Candida species, particularly Candida albicans. There are few classes of antifungal drugs. Although a new class, the echinocandins, has recently been released in the USA and Japan, they are currently only available for intravenous delivery. The drugs of choice for severe candidosis remain the azoles, such as fluconazole (FLC), but C. albicans resistance to these agents can occur in some patient groups. Often resistance is due to energy-dependent drug efflux mediated by plasma membrane proteins such as Cdr1p and Cdr2p. Objectives: The aim of this study was to investigate fungal drug efflux mechanisms in order to devise ways of abrogating efflux-mediated resistance. Methods: Individual alleles of drug efflux pump genes from the diploid fungus C. albicans were cloned and hyper-expressed in a Saccharomyces cerevisiae host strain deleted in endogenous transporters. The following phenotypes in these strains were investigated: susceptibility to azoles; ability to efflux rhodamine 6G (R6G); and pump ATPase activity. Results: Individual C. albicans ATCC 10261 pump alleles (CDR1A, CDR1B, CDR2A, and CDR2B) conferred on the hyper-susceptible S. cerevisiae host different levels of resistance to azole antifungals, and the ability to efflux R6G at different rates. Strains expressing the two CDR2 alleles showed different susceptibilities to FLC, R6G and cerulenin. Analysis of CDR2 alleles from FLC-resistant clinical C. albicans isolates revealed adjacent single nucleotide polymorphisms (G1473A, I1474V) that correlated with altered pump function when expressed in S. cerevisiae. The dependence of pump function on these amino acids was tested by site-directed mutagenesis. Conclusions: We have identified that amino acid residue 1473 in the putative trans-membrane helix 12 of Cdr2p is important for pump function and thus gained an insight into the pumping mechanism. This work was supported by NIH NIDCR grant DE15075.