Based on the initial optimization of DMEM:F12(1:1), two kinds of serum-free media, 11G-SG-SFM and 11G-SE-SFM, were formulated by using orthogonal experiments for the growth of genetically engineered CHO cell line 11G and the production of recombinant prourokinase (pro-UK) in a suspension culture, respectively. Growth of 11G cells in 11G-SG-SFM was comparable with that observed in DMEM:F12 + 5% NBS and CHO-S-SFM. 11G cells grew slowly in 11G-SE-SFM, but secreted a relatively high level of pro-UK. The pro-UK productivity of 11G cells in 11G cells in 11G-SE-SFM reached 800 to 1,000 IU/10(6). d-1 and was about 80% and 20% higher than in DMEM:F12 + 5% NBS and CHO-S-SFM, respectively.