We investigated the fatty acid specificity of 1,3-positional specific Rhizopus delemar lipase in acidolysis using a randomly interesterified oil, of which the constituent fatty acid contents were approximately the same, as a substrate. Acidolysis was carried out at 30 degrees C with shaking in a reaction mixture containing caprylic acid (CA)/the randomly interesterified oil (2:1, w/w) and the lipase immobilized on a ceramic carrier (4% of reaction mixture). The activity on each fatty acid was experimentally determined as the ratio of the content of the fatty acid exchanged for CA to that of the fatty acid existing at the 1(3)-position of the substrate oil, and regarded as the fatty acid specificity in acidolysis. The fatty acid specificity in hydrolysis was investigated by analysis of the composition of fatty acids liberated from the same randomly interesterified oil. The Lipase acted strongly on myristic, palmitic, palmitoleic, stearic, oleic, linoleic, and alpha-linolenic acids, and moderately on arachidonic and eicosapentaenoic acids. The activities on these fatty acids relative to that on oleic acid were almost the same in acidolysis and hydrolysis. On the other hand, the lipase acted moderately on gamma-linolenic and docosahexaenoic acids in the hydrolysis, but only very weakly on these fatty acids in the early stage of acidolysis. This shows that the fatty acid specificity in acidolysis is stricter than that in hydrolysis. Since the kinetics of acidolysis of borage oil, arachidonic acid-containing oil (TGA-25), and tuna oil could be easily predicted from the fatty acid specificity in acidolysis, it was concluded that the specificity was accurately evaluated. In addition, the fatty acids at the 1(3)-position of borage oil and TGA-25 were completely exchanged for CA in a total of three cycles of the reaction, and the contents of fatty acids other than CA in the transesterified oils agreed with that at the 2-position of the oils. Therefore, it was shown that repeated acidolysis using the immobilized 1,3-specific lipase could be applied to regiospecific analysis.