Abstract Regulation of melanin biosynthesis in relation to appressorium differentiation of Colletotrichum lagenarium was investigated. When spores of the parent strain 104-T were incubated at 24°C, appressorial pigmentation started at 6 h of incubation and was preceded by appressorim swelling; appressoria were darkly pigmented at 12 h of incubation. The same time course of appressorial pigmentation was observed in albino mutant 79215 when scytalone, a natural precursor of melanin biosynthesis, was applied before the swelling of appressoria. In accordance with this result, [ 14C]scytalone was not incorporated into germlings of albino mutant 79215 before the swelling of appressoria. Cycloheximide applied 1 h or more after incubation of spores of the parent strain 104-T, or of albino mutant 79215 treated with scytalone, inhibited neither appressorium formation nor appressorial pigmentation. These results indicate that enzymes involved in melanin biosynthesis subsequent to scytalone are preexisting enzymes or synthesized as inactive forms during 1 h of incubation, and that they are activated during appressorium differentiation. In addition, an early step(s) prior to scytalone in the melanin biosynthesis of appressoria was temperature sensitive; when colorless appressoria of the parent strain 104-T formed during 6 h of incubation at 24°C were postincubated at 32°C, the appressoria did not melanize, whereas application of scytalone to the postincubation at 32°C permitted melanization of the appressoria. Also, albino mutant 79215 formed melanized appressoria during postincubation at 32°C in the presence of scytalone. These results indicate that high temperatures inhibit melanin biosynthesis by inhibiting one or more steps prior to scytalone synthesis.