Abstract: Isolated hepatocytes in suspension provide a number of advantages for use in bioartificial liver device, however, poor stability of this cell preparation at physiological temperatures is an apparent barrier preventing their use. We therefore investigated the integrity and differentiated function of isolated rat hepatocytes under conditions of mild hypothermia. Isolated hepatocytes were suspended in a bicarbonate buffered saline medium, supplemented with glucose and bovine serum albumin (BSA), and maintained for 48 h at 25 °C on a rotary shaker under an atmosphere of 95% O2 and 5% CO2. Under these conditions there was no significant decline in cell viability and good preservation of cellular morphology on transmission electron microscopy for at least 24 h. Isolated hepatocytes in suspension at 25 °C were also able to maintain normal Na + and K + ion gradients. The cellular energy status ([ATP], ATP/ADP ratio, cytoplasmic and mitochondrial redox potentials), metabolic function (urea synthesis and ammonia removal), albumin synthesis and phase I and phase II drug detoxification activity of these cells were also maintained for at least 24 h post isolation. These observations demonstrate the robust nature of mildly hypothermic isolated hepatocytes in suspension and encourage further studies re-examining the feasibility of using this cell preparation in bioartificial livers.