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Primary hepatocytes in culture have been reported to lose differentiated function rapidly, thereby limiting their use in any long term studies. The loss in differentiated function is especially noticeable for xenobiotic metabolising activity associated with the cytochrome P-450 enzyme family [l]. In an attempt to circumvent this problem, various culture media, supplementary factors, substrata and co-culturing techniques have been used by different laboratories [2-41. The use of these wide ranging methodologies to maintain differentiated function has made comparison of work conducted by different workers very difficult, and so we have initiated a programme of work aimed at the standardisation of a technique for the maintenance of drug metabolism in hepatocytes. This study describes preliminary work on the maintenance of steroid 16a-hydroxylase activity in rat hepatocytes cultured in different media in the absence and presence of insulin and hydrocortisone.Hepatocytes were isolated from adult, male Wistar rats by a modification of the two step collagenase perfusion method of Seglen [5]. Hepatocytes were only used if their viability on isolation (as measured by trypan blue exclusion) exceeded 90%. Metabolising activity was measured by incubating cells with 14-C androsten$ione (1OOpg) for 30 min. at 37°C.Metabolites were quantified following separation by thin layer chromatography of extracted material [6]. Cells were cultured on NUNCLON 9cm dishes at lo7 cellsldish and kept in an incubator at 37°C in an atmosphere of 5 % CQ/95% air. Initial 24h incubations were conducted in media supplemented with 17.5% serum (2.5% foetal calf serum; 15% horse serum), whilst all further incubations were conducted in serum-free media containing 0.1% BSA. The effects of insulin and/or dexamethasone were investigated by conducting an initial 30 min. preincubation prior to the addition of androstenedione. Table 1.Effects of different media and suDDlementarv factors gn I6~~-hvdroxylase activitv in hepatocvtes maintained in primary culture, Results expressed as percentage of zero time values f sd.