Beside de novo fatty acid synthetase, two kinds of fatty acid elongating systems have been found in Mycobacterium smegmatis; they are the malonyl-CoA-dependent, acyl carrier protein (ACP)-requiring system (fatty acid synthetase II, named by K. Bloch's group, 1969) and the acetyl-CoA-dependent ACP-non-requiring one (acetyl-CoA-dependent elongation of fatty acids reported by us, 1977). Now, a third fatty acid elongating system, malonyl-CoA-dependent and ACP-non-requiring, has been isolated from an extract of this microorganisms, separately from each of the previous two elongating systems. Primer specificity and cofactor requirements, especially of pyridine-nucleotide-coenzyme, of the last elongation system also distinguish it from the two previously known systems. All three systems, however, were found in a soluble fraction of M. smegmatis, therefore the regulatory mechanism for these elongation systems should be investigated hereafter.