The antiretroviral nucleoside reverse-transcriptase inhibitor (NRTI) stavudine (d4T) can induce mild to severe liver injuries such as steatosis (i.e. triglyceride accumulation), steatohepatitis and liver failure. NRTI-induced toxicity has been ascribed to the inhibition of mitochondrial DNA (mtDNA) replication causing mtDNA depletion and respiratory chain dysfunction. This can secondarily impair the tricarboxylic acid cycle and fatty acid oxidation (FAO), thus leading to lactic acidosis and hepatic steatosis. However, NRTIs could also impair mitochondrial function and induce hepatic steatosis through other mechanisms. In this study, we sought to determine whether d4T could inhibit mitochondrial FAO and induce triglyceride accumulation through a mtDNA-independent mechanism. Since human tumoral and non-tumoral hepatic cell lines were unable to efficiently oxidize palmitic acid, the effects of d4T on mitochondrial FAO were assessed on cultured rat hepatocytes. Our results showed that 750 microM of d4T significantly inhibited palmitic acid oxidation after 48 or 72 h of culture, without inducing cell death. Importantly, high concentrations of zidovudine and zalcitabine (two other NRTIs that can induce hepatic steatosis), or beta-aminoisobutyric acid (a d4T metabolite), did not impair FAO in rat hepatocytes. D4T-induced FAO inhibition was observed without mtDNA depletion and lactate production, and was fully prevented with l-carnitine or clofibrate coincubation. l-carnitine also prevented the accretion of neutral lipids within rat hepatocytes. High concentrations of d4T were unable to inhibit FAO on freshly isolated liver mitochondria. Moreover, a microarray analysis was performed to clarify the mechanism whereby d4T can inhibit mitochondrial FAO and induce triglyceride accumulation in rat hepatocytes. The microarray data, confirmed by quantitative real-time PCR analysis, showed that d4T increased the expression of sterol regulatory element-binding protein-1c (SREBP1c) and reduced that of microsomal triglyceride transfer protein (MTP). Finally, d4T-induced alteration of SREBP1c and MTP expression was partially prevented by l-carnitine. Thus, short-term incubation with high concentrations of d4T can rapidly induce accumulation of neutral lipids within rat hepatocytes, which can be fully prevented by l-carnitine. Furthermore, our investigations suggested that lipid accumulation could be the consequence of a dual mechanism, namely a mtDNA-independent impairment of mitochondrial FAO and a reduction of lipid export from the hepatocytes.