A perfusion small-scale bioreactor allowing on-line monitoring of the cell energetic state was developed for free-suspension mammalian cells. The bioreactor was designed to perform in vivo nuclear magnetic resonance (NMR) spectroscopy, which is a noninvasive and nondestructive method that permits the monitoring of intracellular nutrient concentrations, metabolic precursors and intermediates, as well as metabolites and energy shuttles, such as ATP, ADP, and NADPH. The bioreactor was made of a 10-mm NMR tube following a fluidized bed design. Perfusion flow rate allowing for adequate oxygen supply was found to be above 0.79 mL min(-1) for high-density cell suspensions (10(8) cells). Chinese hamster ovary (CHO) cells were studied here as model system. Hydrodynamic studies using coloration/decoloration and residence time distribution measurements were realized to perfect bioreactor design as well as to determine operating conditions bestowing adequate homogeneous mixing and cell retention in the NMR reading zone. In vivo (31)P NMR was performed and demonstrated the small-scale bioreactor platform ability to monitor the cell physiological behavior for 30-min experiments.