With the aim of developing an advanced computer control system for hybridoma cell cultures, on-line monitoring and control strategies for nutrient feeding in a fed-batch culture of a hybridoma cell line were investigated. Good correlation was obtained between the on-line monitoring and the off-line analysis of growth, and between the uptake rates of glucose, oxygen and glutamine, respectively. Using the variables monitored on-line, control of the glucose and glutamine concentrations in fed-batch culture was achieved. The characteristics of the glucose-limited fed-batch culture of hybridoma cells were also investigated. In combination with further research on cell physiology, the results of this study should accelerate the development of an advanced and reliable control system for hybridoma cell cultures.