Alkaline lipase production by Acinetobacter radioresistens was performed in a batch fill-and-draw culture, and the results were compared with a batch culture. In the batch culture, the lipase yield was 18 U/mL, which was restricted by the occurrence of protease forming and excessive foaming as cell growth ceased. Because the formation of lipase was found to be chiefly growth-associated, the fill-and-draw culture, which provided an environment for continuous growth, could surpass the limitation encountered in a batch culture and increase the lipase yield to 30 U/mL. The improvement in the lipase yield was suggested to be caused by an adaptation of the cells to the medium during the repeated culture. Although the increase in the lipase yield was accompanied by a decrease in lipase productivity, the fill-and-draw culture could be a better mode for lipase production.