N-Medium-and long-chain acyl-L-amino acids were enzymatically synthesized from the corresponding L-amino acids and fatty acids using a reverse hydrolysis. Enzymes that are suitable for the synthetic reaction of N-acyl-L-amino acids were screened on the basis of hydrolytic activity toward N-lauroyl-L-glutamic acid as an indicator. Acylase I from pig kidney (EC 3.5.1.14) showed the highest N-acyl-L-amino acid hydrolytic activity among 57 commercially available enzymes tested. Acylase I effectively catalyzed the synthesis of N-lauroyl-L-amino acids except for Nlauroyl-L-proline and N-lauroyl-L-tyrosine in a glycerol-water system. Under the optimized reaction conditions, N-lauroyl-L-arginine and N-lauroyl-L-glutamic acid were obtained in conversions of 82 and 44%, respectively. The equilibrium constants calculated from the conversion obtained were 5. 6, 15.4, 18.0, and 39.4 for the syntheses of N-lauroyl-L-glutamic acid, N α -lauroyl-L-lysine, Nlauroyl-L-glutamine, and N-lauroyl-L-methionine, respectively. NAcyl-L-arginines with myristic acid and palmitic acid as the fatty acid were also synthesized using acylase I.