Lipase-catalyzedo-acylations of N-acyl ethanolamines were investigated in organic solvent. Of 23 lipase (esterase preparations, half of the enzymes catalyzed the myristoylation of N-myristoyl ethanolamine in hexane. Reaction conditions for acylation were investigated using lipases such as Lipase QL from Alcaligenes sp., Lipase PS from Pseudomonas sp., and Lipase AK-20 from Pseudomonas sp. which were selected as lipases with higher activities for o-acylation. The most suitable organic solvents studied for o-acylation were hexane and toluene in the case of myristic acid as the acyl donor and acetone and tetrahydrofuran in the case of vinyl myristate as an acyl donor. The appropriate concentration of water was 0.5-1%; addition of mole water reduced the acylation activity of the enzyme because of the reverse reaction. The reaction rate for acylation was accelerated with the fatty acid vinyl ester as an acyl donor. The effective chain length of the acyl donor for o-acylation of N-myristoyl ethanolamine was around C-14-C-18 alkyl chains in free acids but was C-2-C-14 in vinyl esters. The optimum acyl chain length of N-acyl ethanolamine for myristoylation of N-acyl ethanolamine was altered by the acyl donor and solvent. In the case of racemic 3-hydroxymyristic acid as the free acyl donor for. acylation of N-myristoyl ethanolamine using Lipase QL, the resulting compound was only N-myristoyl-o-3-hyrdroxymyristoyl) ethanolamine and the e.e. value of the resulting compound was 50% of the (R) conformation. Lipase QL proved to be useful for acylation using optically active fatty acid.