The biosynthesis of iso-fatty acids was investigated in the myxobacteria Myxococcus xanthus and Stigmatella aurantiaca by feeding labeled precursors of these fatty acids and by conducting detailed analysis of the fatty acid profiles and fatty acid MS and NMR spectra. These experiments revealed that in S. aurantiaca all iso-even fatty acids are formed via alpha-oxidation of iso-odd fatty acids and not by using valine-derived isobutyryl-CoA.