Corynebacterium lepus was grown in 20-liter batch fermentations with kerosene as the sole carbon source. Critical micelle concentration measurements indicated the production of appreciable quantities of biosurfactants. This surface activity of the culture medium was due to lipids, which were extracted and identified. Samples of C. lepus whole broth were taken during a fermentation and monitored for surface tension, amount of surfactant present, and lipid content. The changes in the surfactant measured correlated with concentration changes of several surface-active lipids. An early dramatic increase in surfactant concentration was attributed to the production of a mixture of corynomycolic acids (beta-hydroxy alpha-branched fatty acids). Surface activity at the end of the fermentation was due to a lipopeptide containing corynomycolic acids plus small amounts of several phospholipids and neutral lipids which were identified by thin-layer chromatography.