Lethal effect of long-chain fatty acids on various species of mycobacteria was examined in a buffer of pH 5.6. Oleic, linoleic and myristic acids were most active against all the test strains. Lauric and palmitic acids were also active, but stearic acid was inactive. The unsaturated fatty acids inhibited markedly the activities of the membrane-bound enzymes (acid phosphatase and tetrazolium reductase) of M. bovis. The free fatty acid fraction of “lysosomal components” separated from the normal guinea pig lungs consisted mainly of palmitic (30%), oleic (30%) and linoleic acids (15%) ; consequently the fraction was active in killing mycobacteria. The mycobactericidal activity of free fatty acids was slight at neutral pH and neutralized by a basic protein, protamine. From these observations, discussions were given on the possible conditions required for fatty acids to be involved in the mechanism of resistance to tuberculous infection.