Mammalian cell culture metabolism is characterized by glucoglutaminolysis, that is, high glucose and glutamine uptake combined with a high rate of lactate and non-essential amino acid secretion. Stress associated with acid neutralization and ammonia accumulation necessitates complex feeding schemes and limits cell densities achieved in fed-batch culture. Conventional and constraint-based metabolic flux analysis has been successfully used to study the metabolic phenotype of mammalian cells in culture, while 13C tracer analysis has been used to study small network models and validate assumptions of metabolism. Large-scale 13C metabolic flux analysis, which is required to improve confidence in the network models and their predictions, remains a major challenge. Advances in both modeling and analytical techniques are bringing this challenge within sight. (C)2009 Elsevier Inc.