Physiological state multiplicity was observed in continuous cu Itu res of the hybridoma cell line ATCC CRL-1606 cultivated in glutamine-limited steady state chemostats. At the same dilution rate (0.04 h(-1)), two physiologically different cultures were obtained which exhibited similar growth rates and viabilities but drastically different cell concentrations (7.36 x 10(5) and 1.36 x 10(6) cells/mL). Metabolic flux analysis conducted using metabolite and gas exchange rate measurements revealed a more efficient culture for the steady state with the higher cell concentration, as measured by the fraction of pyruvate carbon flux shuttled into the TCA cycle for energy generation. The low-efficiency steady state was achieved after innoculation by growing the cells in a nutrient rich environment, first in batch mode followed by a stepwise increase of the dilution rate to its set point at 0.04 h(-1). The high-efficiency steady state was achieved by reducing the dilution rare to progressively lower values to 0.01 h(-1) resulting in conditions of stricter nutrient limitation. The high energetic efficiency attained under such conditions was preserved upon increasing the chemostat dilution rate back to 0.04 h-(1) with a higher nutrient consumption, resulting in approximate doubling of the steady state cell concentration. This metabolic adaptation is unlikely due to favorable genetic mutations and could be implemented for improving cell culture performance by inducing cellular metabolic shifts to more efficient flux distribution patterns.