A two-step enzymatic synthesis process of 4-hydroxyisoleucine is suggested. In the first step, the aldol condensation of acetaldehyde and α-ketobutyrate catalyzed by specific aldolase results in the formation of 4-hydroxy-3-methyl-2-keto-pentanoate (HMKP). In the second step, amination of HMKP by the branched-chain amino acid aminotransferase leads to synthesis of 4-hydroxyisoleucine. An enzyme possessing HMKP aldolase activity (asHPAL) was purified 2500-fold from a crude extract of Arthrobacter simplex strain AKU 626. Sequencing of the asHPAL structural gene showed that the purified enzyme belongs to the HpcH/HpaI aldolase family. The 4-hydroxyisoleucine was synthesized in vitro from acetaldehyde, α-ketobutyrate and l-glutamate using a coupled aldolase/branched-chain amino acid aminotransferase bienzymatic reaction.