As a preliminary step towards characterizing genes encoding ATP-binding cassette (ABC) transporters that confer pleiotropic drug resistance in Aspergillus, we used a PCR-based approach to isolate four DNA fragments corresponding to different ABC type transporter genes. DNA sequencing and Southern blot analysis confirmed that they were distinct genes, which were designated abcA-D. One of these genes, abcD, was cloned and characterized. It was found to have a predicted 1,452-amino acid translation product with a calculated molecular mass of 147,467 kDa. The abcD gene specifies a single transcript of approximately 5.0 kb; there was a two- to six-fold enhancement of mRNA levels following exposure to miconazole, camptothecin, methotrexate, and ethidium bromide.