The coryneform bacterium, Brevibacterium ammoniagenes, contains two structurally related but functionally differentiated type I fatty acid synthases, FAS-A and FAS-B. Isolation of homogeneous preparations of both enzymes was achieved by constructing specific fasA and fasB expression systems. In B. ammoniagenes, insertional mutagenesis of fasB allowed the specific production of enzymatically active FAS-A. The corresponding fasA mutant was not suited for FAS-B purification as the level of this enzyme was extremely low, in the fasA-disruptants. Instead, FAS-B could be efficiently expressed in the heterologous host, Escherichia coli. Using specific antisera against each of the two FAS variants, FAS-A was shown to be the predominant FAS protein in B. ammoniagenes. In contrast the two enzymes are expressed at comparable rates in E. coli even though the same upstream sequences were associated with fasA and fasB, as in B. ammoniagenes. Due to their differential capacities of being activated to the phosphopantetheine-containing holo-enzyme in the heterologous host, only FAS-B but not FAS-A exhibited overall FAS activity when isolated from E. coli. Irrespective of their origin, the purified FAS-A and FAS-B proteins were indistinguishable with respect to their flavin fluorescence, their subunit size and their sucrose density gradient sedimentation characteristics. Nevertheless, the in vitro products of both enzymes differ characteristically: while FAS-A synthesizes mainly the 18-carbon fatty acids oleate and stearate with only traces of palmitate, the major product of FAS-B is palmitic acid. No unsaturated fatty acids are produced by FAS-B. Thus, the two B ammoniogenes type I fatty acid synthases differ, in spite of their very similar overall protein structure, in both their ability to synthesize oleic acid and in their chain-length specificities.