Appressoria of the phytopathogenic fungus Colletotrichum lagenarium contain melanin, which has been implicated as an important factor in the penetration of host plants. A cDNA clone containing the melanin biosynthetic gene encoding scytalone dehydratase (SCD1) from C. lagenarium was identified by hybridization with a heterologous cDNA probe from Magnaporthe grisea. The cDNA clone was used to identify a cosmid containing SCD1 in a genomic library of C. lagenarium, and the nucleotide sequence was determined for both the cDNA and genomic clones. The SCD1 gene contained one open reading frame composed of 188 codons and two deduced introns of 57 and 67 nucleotides. The deduced amino acid sequence of the N-terminal region of SCD1 showed high similarity to the amino acid sequence of scytalone dehydratase from Cochliobolus miyabeanus. A plasmid containing the SCD1 gene transformed the melanin-deficient mutant 9201Y (Sed(-)) to the wild phenotype but did not complement the conditional seytalone dehydratase-deficient mutant C. lagenarium 8015. Genomic DNA analysis indicated that the SCD1 gene is a single locus in C. lagenarium. Transcripts of the SCD1 gene were detected 2 h after the start of conidial germination.