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Purpose: Regular consumption of extra virgin olive oil (EVOO) is associated with a low incidence of atherosclerotic diseases. The phenolic component contributes to the hypolipidemic action of EVOO, although the biochemical mechanisms leading this beneficial outcome are not fully understood. Since liver plays a pivotal role in the whole body lipid homeostasis, we investigated the short-term effects of EVOO extract, with a high phenol content (HPE), on lipid synthesis in primary rat hepatocytes. Refined olive oil extract, with a low phenol content, was used throughout this study as a control. Methods: Olive oil phenols isolated with methanolic extractions were subsequently analyzed by high performance liquid chromatography, electrospray ionization tandem mass spectrometry, and gas chromatography mass spectrometry. Rat hepatocytes were obtained from collagenase perfusion of liver. A colorimetric assay was performed to exclude cytotoxicity of the extracts. Radioenzymatic methods were used in order to investigate hepatic lipid metabolism. Results: HPE, dose- (0.1-50 μg/mL) and time-dependently (0.5-4 h) inhibited both lipogenesis and cholesterogenesis ( n = 6, P < 0.05), as well as triglycerides synthesis ( n = 5, P < 0.05). We showed that these effects are attributable to a short-term modulation by HPE of the key enzymes implicated in the abovementioned pathways ( n = 5, P < 0.05). Conclusions: The decrease in hepatic lipid synthesis may represent a potential mechanism underlying the hypolipidemic effect of EVOO phenols. [ABSTRACT FROM AUTHOR]