Colletotrichum lagenarium is a plant pathogenic fungus, and produces melanin that is an essential factor for appressorial penetration into host tissues. The melanin biosynthesis pathway of C. lagenarium starts with pentaketide synthesis catalyzed by polyketide synthase Pks1p. We previously confirmed that the direct product of Pks1p is 1, 3, 6, 8-tetrahydroxynaphthalene. Thus, melanin biosynthesis in this fungus requires the reduction of 1, 3, 6, 8-tetrahydroxynaphthalene to scytalone. We made a double mutant 9141–144 from the thrI mutant 9141 that lacks the ability to metabolize 1, 3, 8-trihydroxynaphthalene. The double mutant 9141–144 could metabolize neither 1, 3, 6, 8-tetrahydroxynaphthalene nor 1, 3, 8-trihydroxynaphthalene. However melanin production by the double mutant was restored by THRI, indicating that Thrlp can metabolize both compounds in vivo. These results demonstrate that two enzymes, Thrlp and a deduced 1, 3, 6, 8-tetrahydroxynaphthalene-specific reductase, are involved in the first reduction step of the melanin biosynthesis pathway of C. lagenarium.