The lipase-catalyzed synthesis of various dipeptides containing D-amino acid was studied Lipase from porcine pancreas (PPL) was the only lipase which catalyzed the synthesis of Bz-Tyr-D-Ala-NH2 from Bz-L-Tyr-OEt and H-D-Ala-NH2. Peptide yields reached 90% in 3-methyl-3-pentanol containing 5% (v/v) buffer. To examine the S-1 and S＇(1) site specificity of PPL, the syntheses of Bz-L-Tyr-D-aa-X and X-L-aa-D-Ala-NH2 were performed separately. The results are summarized as follows : 1. Among various kinds of D-amino acid derivatives, H-D-Ala-NH2, H-D-Ser-OMe, H-D-Thr-OMe, H-D-His-OMe, and beta-Ala-OMe are good acyl acceptors; 2. L-isomers of the above D-amino acids show similar reactivity for peptide synthesis, but corresponding D-isomers result in better yields; 3. In the case of histidine, only D-isomer displays S＇(1) site substrate specificity; and 4. Besides Bz-L-Tyr-OEt, the N-protected alpha-carboxy ester of L-Ala, L-Arg, L-His, L-Trp, and L-Phe can be used as an acyl donor :