Recombinant Chinese hamster ovary (CHO) cells which produce macrophage colony-stimulating factor (M-CSF) were used as a model to systematically develop the serum-free medium. Pre-weaning strategy combined with experimental design was adopted to develop the serum-free medium for CHO cells in this study. First, CHO cells were adapted to grow in a commercial serum-free medium. Then, these weaned cells were adopted to develop our own serum-free medium using experimental design. Insulin and SerEx had significantly stimulatory influence on M-CSF production (P < 0.05) among the seven screened supplements by use of fractional factorial design. Response surface methodology was consequently applied to find optimal concentrations of insulin and SerEx for M-CSF production. By virtue of the pre-weaning strategy and experimental designs, the development of serum-free medium could be simplified and accelerated. (c) 2006 Elsevier Ltd. All rights reserved.