Chinese hamster ovary (CHO) cells are commonly used in the biotechnology industry to produce recombinant proteins. The development of serum-free media in order to maintain mammalian cell viability and reduce cell death in the absence of serum is highly desirable. Our aim was to find new growth factors that could maintain viability and enhance the proliferation of CHO cells under serum-free conditions. Macrophage colony-stimulating factor (M-CSF)-secreting CHO cells were used as a model cell line to evaluate the stimulatory effects of different compounds, including two growth factors, on cell proliferation. Flow cytometry and fluorescence microscopy were used to analyze the cell cycle and monitor apoptotic nuclei during serum-free culture. Among the tested compounds, insulin and basic fibroblast growth factor (bFGF), each used alone, prevented CHO cells from dying and helped maintain cell viability, but cells were unable to re-enter the cell cycle and proliferate. Simultaneous supplementation with insulin and bFGF synergistically promoted the growth of CHO cells and recombinant M-CSF synthesis in basal DMEM/F12 medium. The mitogenic effects of bFGF and insulin under serum-free conditions were confirmed in the CHO-K1 cell line. These results provide useful information for the design and development of serum-free media as well as for antiapoptotic studies of CHO cells under serum-free conditions. (C) 2009, The Society for Biotechnology, Japan. All rights reserved.