Monoester fraction of rat skin surface lipid has been shown to contain more than trace amounts of branched-long-chain fatty acids (BCFAs) of the iso and anteiso series. These BCFAs are biosynthesized using either branched-chain amino acids (BCAAs) or branched-chain alpha-keto acids (BCKAs), or using both of them as precursor. This study has been carried out to address which precursor, BCAAs or BCKAs in the circulation, are mainly utilized for biosynthesis of BCFAs. Dietary supplement of [14C]-valine and isoleucine-induced sharp rise of serum concentration of these two amino acids and their respective alpha-keto acids, and elevated the levels of related BCFAs and branched-chain fatty alcohols in the monoester fraction. A larger proportion of label in the total skin surface lipid was found in the monoester fraction in which fatty acid and alcohol accounted for approx. 80% of total radioactivity. Incorporation of intravenously administered [14C]-BCAAs and BCKAs into the monoester fraction revealed that BCAAs were far better as precursors than BCKAs for BCFA biosynthesis in rat skin. Among three BCAAs, leucine differed from valine or isoleucine in that this amino acid was primarily utilized for production of straight-chain fatty acids rather than for production of related BCFA.