1,8-Dihydroxynaphthalene (1,8-DHN) was isolated from Cochliobolus heterostrophus pale green mutant M13BL19 ( Alb1 + Brn1 + Sal1 + Pgr1-1 ) as the active substance which restored melanization of HE1AS73 ( Alb1-8 Brn1-2 Sal1-1 Pgr1 + ). HE1AS73 oxidized 1,8-DHN, 1-naphthol, N,N -dimethyl- p -phenylenediamine, tetramethylbenzidine, syringaldazine, and 3-(3,4′-dihydroxyphenyl)alanine, but did not oxidize p -cresol and l -tyrosine. None of these compounds was oxidized by HE5R13 ( Alb1-8 Brn1-2 Sal1-1 Pgr1-1 ). The syringaldazine oxidation activity of HE1AS73 was inhibited by diethyldithiocarbamate, cetyltrimethylammonium bromide, and dithiopyrimidine. These results indicate that the Pgr1 + strain contained p -diphenol oxidase which was absent from the Pgr1 − strain. We consider from these results that p -diphenol oxidase, in which Pgr1 is involved, plays a role in melanin synthesis of C. heterostrophus and 1,8-DHN is a natural substrate of this enzyme.