The presence of the 1,8-dihydroxynaphthalene (DHN) melanin biosynthesis pathway was demonstrated in several sapstain fungi including Ceratocystis and Ophiostoma, using both chemical inhibitors and molecular techniques. The inhibitor compounds tricyclazole and carpropamid effectively reduced pigmentation at low concentrations in all tested fungal species, but also lead to growth inhibition at higher concentrations. The inhibitor cerulenin prevented fungal growth in all tested fungi at all tested concentrations, likely due to its inhibitory effect on another enzyme, the metabolically critical fatty acid synthase. Partial DNA sequences for the gene encoding scytalone dehydratase (SD) were obtained from species of Ceratocystis and Ophiostoma and found to have homology with known respective DHN-SD gene sequences. Sequence analysis of the partial SD amino acid sequences showed greater than 80% similarity among the sapstain species, and corresponded well with known phylogenies of sapstain fungi based on rDNA sequences. Aside from the work carried out on the isolate O. floccosum 387N, this is the first known documentation of the melanin pigmentation pathway used by species of the sapstain fungi Ceratocystis, Leptographium and Ophiostoma. Furthermore, since no fungus has ever been found, to our knowledge, to have more than one melanin synthesis pathway, we can state that these species are likely only to use the DHN pathway for melanin production.