Scytalone dehydratase is involved in the production of fungal dihydroxynaphthalene (DHN) melanin. We have isolated and characterized OSD1, a gene encoding scytalone dehydratase from the sap-staining fungus Ophiostoma floccosum by PCR-based cloning. Sequence analysis suggests that the OSD1 gene encodes a protein of 216 amino acids with a molecular weight of 24.2 kDa that shows 51-70% sequence identity to other scytalone dehydratases. The cloned OSD1 contains two introns of 76 bp and 63 bp in length, and is the longest scytalone dehydratase gene sequence so far reported. Transformation of a DHN melanin-deficient, non-pathogenic, mutant of Colletotrichum lagenarium with the OSD1 gene restored melanin production and pathogenicity. The ability of the mutant to produce the OSD1 gene product was confirmed by RT-PCR analysis. These data show that the cloned OSD1 gene product can function in the DHN melanin biosynthetic pathway in C. lagenarium.