A single-copy gene, designated TruMDR2, encoding an ATP-binding cassette (ABC) transporter was cloned and sequenced from the dermatophyte Trichophyton rubrum. The ORF of TruMDR2 was 4048 nt and the deduced amino acid sequence showed high homology with ABC transporters involved in drug efflux in other fungi. The encoded ABC protein predicted 12 transmembrane segments (TMSs) and two almost identical nucleotide-binding domains (NBDs) arranged in two halves in a (TMS6–NBD)2 configuration and could be classified as a member of the multidrug-resistance (MDR) class of ABC transporters. Northern blot analyses revealed an increased level of transcription of the TruMDR2 gene when mycelium was exposed to acriflavine, benomyl, ethidium bromide, ketoconazole, chloramphenicol, griseofulvin, fluconazole, imazalil, itraconazole, methotrexate, 4-nitroquinoline N-oxide (4NQO) or tioconazole. Disruption of the TruMDR2 gene rendered the mutant more sensitive to terbinafine, 4NQO and ethidium bromide than the control strain, suggesting that this transporter plays a role in modulating drug susceptibility in T. rubrum.