Chemoenzymatic synthesis of 1-O-hexadecyl-2-O-palmitoyl-sn-glycerol was achieved by esterification of 1-O-hexadecyl-sn-glycerol, with palmitic acid in the presence of N,N-dicyclohexylcarbodiimide, and then subjected to alcoholysis catalysed by an immobilized 1,3-specific lipase. The highest yield (90% from 0.3 mM) was obtained in 3 h, using methyl isobutyl ketone as solvent with water activity 0.2.