Docosahexaenoic acid (DHA) has attracted attention because of its physiological effects in prevention and treatment of cardiovascular diseases. The concentration of DHA in glyceride is increased by hydrolysis of DHA-rich tuna oil with Candida rugosa. In the present study, hydrolysis of tuna oil by the lipase was carried out at 40degreesC with three kinds of 0.1 M buffer, phosphate buffer, citric acid-phosphate buffer and maleic acid-Tris-NaOH buffer, and the effects of the initial pH of the water phase and the kind of buffer on the activity and specificity of Candida rugosa lipase were studied. When the buffers were used in place of the conventional water, a hydrolysis ratio of tuna oil of more than 85% was obtained, In case of hydrolysis of tuna oil with phosphate buffer of initial pH 6.8, DHA concentration in glyceride at 28 hours of reaction time reached 56% (an almost threefold increase). The glyceride composition of the hydrolyzed oil was predominantly DHA-rich triglyceride and diglyceride, with a very low content of monoglyceride.