The investigation was carried out to identify the active constituent in yeast extract for fruit body formation of Lentinula edodes. The result suggests that free thiamin which is hewn as the active substance for the fruiting of L edodes, was detected but thiamin mono, di, three phosphates were not detected in the yeast extract produced by Difco Co.. Therefore, the thiamin content of the yeast extract was determined, the yeast extract was fractionated to five portion by the post-column fluorescence method. The content of thiamin in yeast extract( 1g) was 0.436mg as thiamin hydrochloride. It was found that 76% of the total thiamin(0.332mg) was contained in fraction II. About 20% of the total thiamin(0.087mg) was present in fraction I, but not in fractions III, IV and V. In accordance with the contents of thiamin in the fractions, the fruit body formation was the highest by the treatment of fraction II(100%) and followed by fraction I (60%), V(50%), III(30%). Thiamin did not influence for the vegetative mycelial growth of L. edodes, but be used for fruit body formation.