AbstractDiglyceride (DG) was prepared by reaction of hydrogenated beef tallow and glycerol in the presence of aPsesudomonas lipase. The yield of DG depended strongly on the reaction temperature. After initial incubation at 60°C for 2 h, followed by the first temperature shift down to 55°C for 4 h and then the second shift down to 48°C for up to 3 d, the reaction mixture became solid and a yield of approximately 90% DG was obtained. About 95% of total DG was 1,3‐DG. The yield of DG was also dependent on the glycerol (GL) to triglyceride (TG) molar ratio. At the molar ratio of 1∶2 (GL/TG), the enzyme‐catalyzed reaction was highly efficient and utilized essentially all of the glycerol. The free fatty acid (FFA) content at equilibrium depended on the water concentration in the glycerol phase. The initial rate of FFA formation was low and was hardly affected by the moisture content between 0.5 and 4%, but, at higher water content (4–6.7%), there was a small increase in the rate.