Substrate limited fed batch cultures were used to study growth and overflow metabolism in hybridoma cells. A glucose limited fed batch, a glutamine limited fed batch, and a combined glucose and glutamine limited fed batch culture were compared with batch cultures. In all cultures mu reaches its maximum early during growth and decreases thereafter so that no exponential growth occurs. Neither glutamine nor glucose seemed growth rate limiting, although the glutamine concentration (>0.085 mM) was lower than reported K-s values and glucose was below 0.9 mM; but some other nutrient(s) was the cause as verified by simulations. Slightly more cells and antibodies were produced in the combined fed batch compared with the batch culture. The specific rates for consumption of glucose and glutamine were dramatically influenced in fed batch cultures resulting in major metabolic changes. Glucose limitation decreased lactate formation but increased glutamine consumption and ammonium formation. Glutamine limitation decreased ammonium and alanine formation, glucose consumption, and lactate formation. The formation of lactate, alanine, and ammonium was negligible in the dual-substrate limited fed batch culture. The efficiency of the energy metabolism increased, as judged by the increase in the cellular yield coefficient for glucose by 100% and for glutamine by 150% and by the change in the metabolic ratios lac/glc, ala/gln, and NHx/gln, in the combined fed batch culture. The data indicate that a larger proportion of consumed glutamine enters the TCA cycle through the glutamate dehydrogenase pathway, which releases more energy from glutamine than the transamination pathway. We suggest that the main reasons for these changes are decreased uptake rates of glucose and glutamine, which in turn lead to a reduction of the pyruvate pool and a restriction of the flux through glutaminase and lactate dehydrogenase. There appears to be potential for further cell growth in the dual-substrate-limited fed batch culture as judged by a comparison of mu in the different cultures.