Bacillus licheniformis JF-2 synthesizes a surfactin-like lipopeptide that is the most effective biosurfactant known. In shake-flask cultures the biosurfactant is produced by actively growing cells (mid-linear phase), but subsequently it becomes rapidly internalized by the cells as soon as the culture enters the stationary phase. This deactivation phenomenon is a major hurdle in the efficient production of the biosurfactant. We have shown that the synthesis of the JF-2 lipopeptide is strongly dependent on O₂ concentration with substantial production observed only in cultures grown under O₂-limiting conditions. In continuous cultures the biosurfactant was produced only within a narrow window of low dilution rates. At a dilution rate of 0.12 h⁻¹ and low dissolved O₂, the biosurfactant concentration was maintained at 33 mg/l, which is virtually the same as the maximum concentration obtained in optimized batch fermentations.