PER.C6 cells were cultivated for propagation of a replication-defective adenovirus vector in serum-free suspension bioreactors. Cellular metabolism during cell growth and adenovirus propagation was fully characterized using on-line and off-line methods. The energy metabolism was found to accelerate transiently after adenovirus infection with increases in glucose and oxygen consumption rates. Similar to other mammalian cells, glucose utilization was highly inefficient and a high lactate:glucose yield was observed, both before and after virus infection. A higher consumption of most of the essential amino acids was observed transiently after the infection, likely due to increased protein synthesis requirements for virus propagation. To improve virus propagation, a medium exchange strategy was implemented to increase PER.C6 cell concentration for infection. During cell growth, a 50% increase in glucose consumption and lactate production rates was observed after initiation of the medium exchange in comparison to the batch phase. This decrease in medium capacity only affected the central carbon metabolism and no increase in amino acid consumption was observed. In addition, even though cell concentrations of up to 10 × 106 cells/mL were reproducibly obtained by medium exchange, infections at cell concentrations higher than 1 × 106 cells/mL did not proportionally improve volumetric adenovirus productivities. No measured nutrient limitation was observed at those high cell concentrations, indicating that adenovirus cell-specific productivity at higher cell concentrations is highly dependent on cell physiology. These results provide a better understanding of PER.C6 cellular metabolism and a basis for intensifying PER.C6 growth and adenovirus propagation. © 2005 Wiley Periodicals, Inc.