Using Crammer's concentration method and paper partition chromatography, several fluorescent substances were obtained from the acetone powder of Mycobacterial cells. These fluorescent substances were purified into single components, and their chemical structures and biological significances were attempted to determine. The results obtained are as follows : 1) Six kinds of fluorescent substances were detected with certaincy from Mycobacterium avium. The red fluorescent substance is coproporphyrin ; the two yellow fluorescent substances are FAD and FMN ; the two bluish green fluorescent substances are both folic acid derivatives, the one has growth promoting activity of Leuconostoc citrovorum but has little activity of Coenzyme F, and the other is the reverse ; the bluish violet fluoresnt substance is an unknown compound. 2) Any difference of distribution of these fluorescent substances were not observed between the resistant strains of both streptomycin and p-aminosalicylic acid and the sensitive strains of them. The red fluorescent substance was not detected specifically from the resistant strain of INAH. This phenomenon suggested us the nature of the resistance and the antibacterial mechanism of INAH. 3) Making clear the distribution of the fluorescent substances, it may be able to classify Mycobacteria.