Proton NMR spectroscopy of biological fluids has produced interesting results lately. We used the technique to investigate the effects of cryopreservation on primary porcine hepatocytes as successful cryopreservation of primary porcine hepatocytes is of importance to the development of bioartificial liver support systems. After isolation 108 hepatocytes were cryopreserved for 1 week in Williams E/10% DMSO, either by quick freezing (−5 to −30 °C/min), slow freezing (−0.3 to −3 °C/min) or stepwise freezing protocols on cell suspensions and confluent cell plates. Plating efficiency was assessed by percentage LDH release. Metabolic functions of cryopreserved hepatocytes at 24 h post-thawing were compared with those of fresh hepatocyte cultures at 48 h. 1H nuclear magnetic resonance spectroscopy of the culture medium post-incubation, using the presaturation technique, assessed the following: glucose metabolism, transamination and glutamine synthesis and succinate synthesis. Freshly isolated cells had a viability of 82 ± 4.3% and a plating efficiency of 87 ± 3.8%. All cryopreservation protocols resulted in significantly reduced viability and plating efficiency. No significant differences were observed between different cryopreservation media or protocols. When comparing cryopreserved with freshly isolated cells, we observed that metabolism of acetyl-CoA precursors was significantly impaired in cryopreserved cells. Lactate and pyruvate production was also significantly less, although glucose consumption was similar. No differences were observed in gluconeogenic amino acid metabolism, transamination and urea synthesis. 1H NMR spectroscopy can be used to provide information about metabolic activity and functions of cultured primary cells. Copyright © 2002 John Wiley & Sons, Ltd.