Diacylglycerols (DAG) are important intermediates in lipase-catalyzed interesterification, but a high DAG concentration in the reaction mixture results in a high DAG content in the final product. We have previously shown that a high DAG concentration in the reaction mixture increases the degree of acyl migration, thus adding to the formation of by-products. In the present study we examined the influence of water content, reaction temperature, enzyme load, substrate molar ratio (oil/capric acid), and reaction time on the formation of DAG in batch reactors. We used response surface methodology (RSM) to minimize the numbers of experiments. The DAG content of the product was dependent on all parameters examined except reaction time. DAG formation increased with increasing water content, enzyme load, reaction temperature, and substrate ratio. The content of sn-1,3-DAG was higher than that of sn-1,2-DAG under all conditions tested, and the ratio between the contents of the former compounds and the latter increased with increasing temperature and reaction time. The water content, enzyme load, and substrate ratio had no significant effect on this ratio. The DAG content was positively correlated with both the incorporation of acyl donors and the degree of acyl migration.