NMR feasibility was established for a coaxial hydrophobic-membrane bioreactor containing isolated rat hepatocytes with features designed to mimic the human liver. A novel triple-tuned NMR probe and a perfusion system controlling temperature, gas concentrations, flow-rate, and pH were used. We determined the optimum coaxial interfiber distance (i.e. diffusion distance) for maintaining hepatocyte viability in two bioreactor prototypes. Prototype no. 1 and no. 2 had diffusion distances of 500 microns and 200 microns, respectively. Cell viability was established by 31P NMR and trypan blue exclusion. Only prototype no. 2 maintained cell viability for more than 6 h, indicating the importance of diffusion distance. 31P spectra obtained over this 6 h time period were similar to in vivo spectra of rat liver. The 31P spectra were found to be more sensitive to subacute cell viability than trypan blue exclusion. In the 1H and 31P spectra, 1H2O and inorganic phosphate signals were split in two at all flow-rates, probably due to bulk magnetic susceptibility effects originating from the three bioreactor compartments. MRI was useful for quality control and determining flow dynamics, fiber integrity, and cell inoculate distribution. MRI revealed that the inner fibers were not centered in either prototype. Although an increased flow-rate did not influence spectral resolution or chemical shifts, significant degradation of MRI quality occurred above 50 mL/min. NMR spectroscopy and imaging provide valuable, real-time information on cell biochemistry and flow dynamics which can be used in development and monitoring of bioreactors designed as artificial livers.