Fonsecaea pedrosoi, a dematiaceous fungus, is the main agent responsible for chromoblastomycosis, a chronic and progressive mycosis of the skin and subcutaneous tissues. This disease can cause different types of lesions depending on the immune status of the host. Its treatment is complicated by the toxicity of available antifungal agents as well as drug resistance. In this work, an ATP-binding cassette (ABC) transporter in this fungus was characterised, with the degree of expression related to the drug resistance of two strains (a patient isolated strain and a laboratory strain). A 150 kDa protein was detected by western blotting. The ATPase activity of membrane preparations was also evaluated. The F. pedrosoi transporter appears to behave like Pdr5p, a well-studied multidrug resistance transporter in Saccharomyces cerevisiae with the ability to hydrolyse different triphosphate nucleotides, as well as its response to classical inhibitors tested. Finally, a reverse transcription polymerase chain reaction (RT-PCR) approach was used and a 400 bp product was detected, corresponding to the highly conserved ATP-binding domain of ABC transporters. We suggest that an ABC transporter must be involved in F. pedrosoi multidrug resistance, and a complete understanding of this protein could bring an important contribution to antifungal treatment of this disease.