A biotransformation process using Mycobacterium sp was studied for androsta-1, 4-diene-3,17-dione (ADD) and androsta-4-ene-3,17-dione (AD) production from cholesterol. Cholesterol has a poor solubility in water (similar to1.8mg dm(-3) at 25degreesC), which makes it difficult to use as the substrate for biotransformation. Lecithin is a mixture of phospholipids of phosphatidylcholine (PC) and phosphatidylethanolamine (PE), which behave like surfactants and can form planar bi-layer structures in an aqueous medium. Therefore, a small amount of lecithin (<1g dm(-3)) can be used to form stable colloids with cholesterol at a relatively high concentration (20 g dm(-3)) in water. In this work, an energy density of 1000J cm(-3) from sonication was provided to overcome the self-association of cholesterol and to generate a stable lecithin-cholesterol suspension that could be used for enhanced biotransformation. The lecithin-cholesterol suspension was stable and could withstand typical autoclaving conditions (121 degreesC, 15 psig, 20 min). In contrast to conventional surfactants, such as Tween 80, that are commonly used to help solubilize cholesterol, lecithin did not change the surface tension of the aqueous solution nor cause any significant foaming problem. Lecithin was also biocompatible and showed no adverse effect on cell growth. Compared with the medium with Tween 80 as the cholesterol-solubilizing agent, lecithin greatly improved the biotransformation process in regard to its final product yield (similar to59% w/w), productivity (0.127-0.346 g dm(-3) day(-1)), ADD/AD ratio (6.7-8), as well as the long-term process stability. Cells can be reused in repeated batch fermentations for up to seven consecutive batches, but then lose their bioactivity due to aging problems, possibly caused by product inhibition and nutrient depletion.