A collection of Aspergillus fumigatus mutants highly resistant to itraconazole (RIT) at 100 micro g ml(-1) were selected in vitro (following UV irradiation as a preliminary step) to investigate mechanisms of drug resistance in this clinically important pathogen. Eight of the RIT mutants were found to have a mutation at Gly54 (G54E, -K, or -R) in the azole target gene CYP51A. Primers designed for highly conserved regions of multidrug resistance (MDR) pumps were used in reverse transcriptase PCR amplification reactions to identify novel genes encoding potential MDR efflux pumps in A. fumigatus. Two genes, AfuMDR3 and AfuMDR4, showed prominent changes in expression levels in many RIT mutants and were characterized in more detail. Analysis of the deduced amino acid sequence encoded by AfuMDR3 revealed high similarity to major facilitator superfamily transporters, while AfuMDR4 was a typical member of the ATP-binding cassette superfamily. Real-time quantitative PCR with molecular beacon probes was used to assess expression levels of AfuMDR3 and AfuMDR4. Most RIT mutants showed either constitutive high-level expression of both genes or induction of expression upon exposure to itraconazole. Our results suggest that overexpression of one or both of these newly identified drug efflux pump genes of A. fumigatus and/or selection of drug target site mutations are linked to high-level itraconazole resistance and are mechanistic considerations for the emergence of clinical resistance to itraconazole.