Branched chain fatty acids (BCFAs) of the iso and anteiso series are synthesized from branched-chain amino acids (BCAAs) in many bacteria and in animal skin. This communication describes the biosynthesis of BCFA in Bacillus subtilis and rat skin. BCFAs of Bacillus subtilis are synthesized from the α-keto acids of BCAAs. Two distinct decarboxylases were present in the crude BCFA synthetase of Bacillus subtilis : branched chain α-keto acid (BCKA) decarbaxylase and pyruvate decarboxylase. BCFA synthetase activity was completely lost upon immunoprecipitation of BCKA decarboxylase, suggesting that this enzyme is essential for BCFA biosynthesis by Bacillus subtilis . A BCKA decarboxylase-mediated mechanism was postulated for BCFA biosynthesis. Supplementation of the diet with valine and isoleucine increased the levels of respective BCFAs in the skin surface lipids of rat. However, this was not the case for leucine. Serum levels of valine, isoleucine and their α-keto acids correlated well with the level of the respective BCFAs in the skin surface lipids. Incorporation in vivo of [14C] -BCAA and BCKA into the skin surface lipids revealed that valine and isoleucine rather than their α-keto acids were used mainly for BCFA biosynthesis by rat skin. BCFA synthesis by rat skin differed from that by bacteria in that the systems did not use leucine as a primer. Leucine was used for biosynthesis of straight-chain fatty acids rather than for BCFAs. Substrate specificity and stereoselectivity in vivo of animal BCFA synthesis was described.