The multienzyme complex from Mycobacterium phlei which catalyzes the synthesis of long chain fatty acids from acetyl-CoA and malonyl-CoA requires a heat-stable fraction (stimulating factor, SF) for activity. Fractionation of heat-treated M. phlei extracts affords two stimulatory subfractions, one of which (SF2) can be replaced by FMN. The other (SF1) is further separable into 3 polysaccharides (PSI, PSII, and PSIII). PS, contains about 95% 3-0-methylmannose and 5% mannose; the sugar composition of PSI, and PSI,, is about 55% 6-0-methylglucose and 45% glucose for both. Each of the three purified polysaccharides, in combination with FMN, substitutes for the crude stimulating factor. The polysaccharides exert their effect on the fatty acid synthetase by lowering the Km for acetyl-CoA about 50-fold.