The development of m vitro models for toxicity testing of food components is dependent on the availability of standardized methods of cell isolation, preparation and culture. Many groups isolate and use rat hepatocytes for a variety of purposes but the methods used for isolation vary greatly Differences in isolation technique have been identified as a possible source of variation in the results obtained. This issue has led the European Centre for the Validation of Alternative Methods (ECVAM), following discussion with experts in the field of hepatocyte research, to recommend a standard hepatocyte isolation procedure. This project focused on the validation of a cell model consisting of short-term suspension cultures of rat hepatocytes isolated using the method recommended by ECVAM Characterisation of the hepatocytes with respect to structural and biochemical integrity and the retention of liver function was carried out via the assessment of dye uptake, cytosolic enzyme leakage, DNA, protein and ATP content, gluconeogenesis, cholesterogenesis and protein synthesis Hepatocytes were found to be structurally and biochemically intact and to retain liver function. The effects of four micro-food components, 7-ketocholesterol, cholestanetriol, Covi-ox and conjugated linoleic acid, on biochemical functionality and on the antioxidant defense system were then studied. The antioxidant defense system was studied through assessment of the extent of lipid peroxidation and the activity of the enzymes superoxide dismutase, catalase and glutathione peroxidase 7-Ketocholesterol and cholestanetriol increased the activity of some or all of the antioxidant enzymes measured but did not induce lipid peroxidation Coincubation of oxysterols with either Covi-ox or CLA decreased the activity of the antioxidant enzymes to levels close to or below control Lipid peroxidation in cells treated with CLA or Covi-ox alone or in combination with 7-ketocholesterol or cholestanetriol was not found to be significantly different to control. This study provides indirect evidence that none of the test substances, at the concentrations studied, exerted prooxidant effects such as to induce lipid peroxidation in rat hepatocytes.