Rapid assessment of the cells physiological state during a culture is essential for bioprocess optimization and the design of effective control strategies. In this work, an approach was developed to provide an on-line estimation of the intracellular flux distribution of cells grown in perfusion cultures, based on a comprehensive metabolic network involving 40 biochemical reactions and 46 components. The specific uptake and production rates were evaluated from daily nutrient and metabolite concentration measurements, using an extended Kalman-filter for noise reduction and rate estimation. The biosynthetic rates were determined from an on-line estimate of the specific growth rate using a recursive least-squares method. Our results demonstrate that this approach allows monitoring of the cells metabolic activity and can be a useful tool to facilitate process development. (c) 2006 Elsevier Ltd. All rights reserved.