Abstract Hepatocyte cryopreservation is essential to ensure a ready supply of cells for use in transplantation or as part of an extracorporeal liver assist device to provide on-demand liver support. To date, most of the work on hepatocyte cryopreservation has been performed on isolated hepatocytes, and has generally yielded cells which display low viability and greatly reduced short-term function. This report presents the development of a freezing procedure for hepatocytes cultured in a sandwich configuration. A specially designed freezing unit was used to provide controlled temperatures throughout the freeze-thaw cycle. Cooling rate, warming rate, and final freezing temperature were evaluated as to their effect on hepatocyte function as judged by albumin secretion. Under optimized conditions (cooling at 5°C/min and warming at ≥400°C/min), freezing to −40°C resulted in full recovery of albumin secretion within 2–3 days postfreezing, whereafter albumin secretion levels remained normal for the duration of the experiments (2 wks). Freezing to −80°C lead to an approximate 70% recovery of long-term protein secretion when compared to control cultures. In addition, the overall hepatocyte morphology as judged by light microscopy, closely followed the functional results. The sandwich culture configuration, thus, enables hepatocytes to maintain a satisfactory level of long-term protein secretion after a freeze—thaw cycle under optimized conditions, and offers an attractive tool for further studies into the mechanisms of freezing injury and subsequent hepatocellular recovery. These results are a promising step in the development of satisfactory storage procedures for hepatocytes.