Hepatic steatosis is an important risk factor for the development of inflammation, fibrosis and impaired liver regeneration. The factors regulating lipid accumulation and driving hepatic steatosis toward inflammation, fibrosis and impaired regeneration are largely unknown. The aim of this study was to identify major alterations in gene expression occurring in steatotic hepatocytes, and to analyze how these changes impact cellular processes associated with steatosis. Microarray gene chips and RT-PCR were performed to analyze changes in gene expression induced in fatty human immortalized hepatocytes after treatment with 50 muM oleic acid for 7 days. Lipid metabolism and triglyceride accumulation in these cells was examined by Oil-Red-O staining, thin-layer chromatography (TLC) and immunofluorescence. Caspase 3 activity, BrdU incorporation and trypan blue exclusion were used to study apoptosis, proliferation and cell viability. Finally, quantitative analysis of signalling induced by insulin was performed by Western blot. Characterization of steatosis in three hepatocyte-derived cell lines indicated that the immortalized human hepatocytes (IHH) line was the most appropriate cell line for this study. Gene expression analysis showed significant alterations in the transcription of two major classes of genes involved either in cholesterol and fatty acid biosynthesis, as well as lipid export, or in apoptosis and cell proliferation. Such changes were functionally relevant, since TLC indicated that synthesis and accumulation of triglycerides were increased in steatotic cells, while synthesis of cholesterol and fatty acids were decreased. Lipid accumulation in IHH was associated with an increased apoptosis and an inhibition of cell proliferation and viability. No detectable changes in genes associated with insulin resistance were observed in steatotic cells, but signalling induced by insulin was more efficient in steatotic IHH as compared to control cells. We conclude that IHH represent a new valuable model of steatosis, not associated with insulin resistance, to study at both the genetic and functional level factors involved in the process of lipid accumulation and steatosis-associated liver injury.