Understanding the regulation of hepatocyte lipid metabolism is important for several biotechnological applications involving liver cells. During exposure of hepatocytes to plasma, as is the case in extracorporeal bioartificial liver assist devices, it has been reported that hepatic-specific functions, e.g., albumin and urea synthesis and diazepam removal, are dramatically compromised and hepatocytes progressively accumulate cytoplasmic lipid droplets. We hypothesized that the composition of hepatocyte culture medium significantly affects lipid metabolism during subsequent plasma exposure. Rat hepatocytes we-re cultured in medium containing either physiological (50 muU/mL) or supraphysiological (500 mU/mL) insulin levels for 1 week and then exposed to human plasma supplemented with or without amino acids. We found that insulin's anabolic effects, such as stimulation of triglyceride storage, were carried over from the pre-conditioning to the plasma exposure period. While hepatocytes cultured in high insulin medium accumulated large quantities of triglycerides during subsequent plasma exposure, culture in low insulin medium largely prevented lipid accumulation. Urea and albumin secretion, as well as the ammonia removal rate, were largely unaffected by insulin but increased with amino acid supplementation. Thus, hepatocyte metabolism during plasma exposure can be modulated by medium pre-conditioning and supplements added to plasma.