Two unnatural amino acids were found as by-products in isoleucine fermentation from threonine by Serratia marcescens. These amino acids were identified as L-norvaline (2-aminopentanoic acid) and L-homoisoleucine (2-amino-4-methylhexanoic acid). Formation of L-norvaline and L-homoisoleucine was not observed when L-leucine was added to the medium. The leucine auxotroph derived from the isoleucine accumlator did not produce L-norvaline or L-homoisoleucine even during the accumulation of large amounts of isoleucine. It is suggested that L-norvaline and L-homoisoleucine formation is closely related to leucine biosynthesis.