The Mycobacteria contain large amounts of lipids and several of these lipids are biologically active. Fatty acids that are utilized for lipid synthesis are synthesized from 2C-unit as acetate or malonate through the malonyl CoA pathway. In the present report, the quantitative and qualitative changes of activity for the fatty acid synthesis were studied at the varying age of culture. The studies on the activities of the cell-free extracts of H37Ra strain cultured for 2, 3, 4 and 5 weeks for the incorporation of acetate or malonate carbon into long chain fatty acids were carried out. 1. In the case of either acetate or malonate for substrate, the highest activity was observed in the cell-free extract taken from the Mycobacteria cultured for 2 weeks. The activity was reduced for farther duration of culture over 2 weeks. 2. The distribution of radioactivity incorporated into long chain fatty acids from either acetate-1-(14)C or malonate-2-(14)C was checked with gas liquid chromatography. At any age of culture, the incorporation into hexacosanoic acid was the highest. And the incorporation rate into hexacosanoic acid and fatty acids of fewer than 26 carbons was decreased with prolonged culture, but the incorporation rate into fatty acids of more than 26 carbons was increased with the prolongation of culture age.