We have previously shown [Sakai et al., J. Biosci. Bioeng., 88, 306-309 (1999)] that exogenously supplied phosphatidic acid (PA) and lysophosphatidic acid (LPA) promoted the growth of Chinese hamster ovary (CHO) cells in serum-free culture. However, the direct addition of high concentrations of these phospholipids alone to the culture medium resulted in the formation of precipitates. We therefore examined the use of two nonionic surfactants, Tween 80 and Pluronic F-68, as a means of supplying PA more effectively to CHO cells in a serum-free culture. A clear dispersion of PA from egg yolk lecithin that could be successfully sterile-filtered was obtained by using Tween 80 or Pluronic F-68. When PA prepared with either of the surfactants was added to serum-free media, precipitation was noticeably reduced. Furthermore, the growth-promoting activity of PA was considerably enhanced by the presence of the surfactants. Since Tween 80 and Pluronic F-68 themselves possessed no growth-stimulating property, it was suggested that the enhanced growth-promoting activity results from the improved availability of PA to the cells. The use of Tween 80 with PA analogues having saturated acyl chains also accelerated cell growth, whereas these PAs showed little growth-promoting activity, due to their poor water-solubility, when added alone.