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ABSTRACT. The objective of this study was to determine the effects of NEFA and glucose on carnitine palmitoyltransferase-I (CPT-I) mRNA expression in cultured bovine hepatocytes using real-time reverse transcription polymerase chain reaction and ELISA methods. The results indicated that CPT-I transcription increased gradually, but that CPT-I translation was not significantly changed, with glucose concentrations ranging from 0 to 3.0 mmol/L (P<0.01). Furthermore CPT-I transcription and translation were enhanced significantly when the NEFA concentrations increased from 0 to 1.2 mmol/L and decreased significantly when the NEFA concentrations increased from 1.2 to 4.8 mmol/L (P<0.01). A high concentration NEFA was found to reduce fatty acid oxidation, potentially explaining the development from NEB to ketosis in dairy cows. The main physiological characteristic of peripartum cows is negative energy balance (NEB) [5]. Dairy cows display a relatively low concentration of blood glucose and concurrent high levels of non-esterified fatty acids (NEFA) in the initial stage of NEB [12]. Elevated blood NEFA and low blood glucose are the two key characteristics responsible for the initial serology of NEB [16], and these factors may have an important influence on the development of NEB. Complete oxidation of fatty acids (FAs) in the liver can improve metabolic processes and provide more ATP to relieve NEB and diminish the ketone concentration in blood or triacylglycerol accumulation in the liver. Therefore, the oxidation metabolism pathway of NEFA may be the main pathway providing energy during NEB and concurrent low blood glucose [2,9]. The transfer of activated FAs (acyl-CoA) into mitochondria is controlled by the enzyme carnitine palmitoyltransferase-I (CPT-I). This step positively correlates with the velocity of transfer of acyl-CoA into mitochondria [6] and controls the process of FA metabolism.The objective of this study was to detect the effect of NEFA and glucose on CPT-I mRNA expression and translation levels in bovine hepatocytes in vitro. Our findings provide an insight into how the two initial serology factors of NEB, namely a high NEFA concentration and low blood glucose level, affect FA oxidation metabolism.Hepatocyte cells were cultured as described previously [7,19], with some modifications to optimize the procedures for bovine liver cells. Hepatocytes were isolated from an excised lobe and plated as described previously [15], with some modifications. The cultured cells were cultured in sixwell plates at a density of 10 6 cells/cm 2 (Costar, Corning Incorporated, Corning, NY, U.S.A.). After a 4-hr attachment period, the media and unattached cells were aspirated and replaced with RPMI 1640 culture medium containing 10% fetal bovine serum. The cells were then cultured at 37C in 5% CO 2 . Every 24 hr, the media were aspirated and refreshed [4,18]. At 72 hr, liver cells was cultured with glucose-and NEFA-free RPMI 1640 culture medium. NEFA and glucose were added to the media separately [15].