The influence of the shear rate, the membrane pore size, and the age of the culture at time of harvest on transmembrane pressure (TMP) increase and membrane fouling during the microfiltration of a Chinese Hamster Ovary (CHO) cell culture supernatant was investigated. A hollow fiber microfiltration system operated at constant permeate flux was used. The highest TMP increase with filtration time was observed for the small membrane pore Size (0.20 mu m) operated at the higher shear rate (8.000 s(-1)). Furthermore, the high overall fouling observed with the small membrane pore size was also associated with the highest irreversible fouling and the most significant decrease of predicted open pore area. The predicted reduction in open pore area obtained with the combined pore blockage and cake formation mechanism could explain the observed TMP profiles. Based on the overall membrane fouling, the long-term irreversible fouling and the initial fouling rate, derived from oil empirical curve fitting, no effect of the time of harvest was observed but a dependence of the initial fouling rate on the shear rate was identified. Treatment of the fouled membrane with water showed the presence of a more significant reversible fouling at high shear rates and increased irreversible fouling with smaller membrane pore Size. It is recommended to use the large membrane pore Size (0.45 mu m) and the low shear rate (4,000 s(-1)) to minimize fouling associated with the soluble components of serum-free CHO cell culture supernatant.