Accumulation of intracellular lipid in obesity is associated with metabolic disease in many tissues including liver. Storage of fatty acid as triglyceride (TG) requires the activation of fatty acids to long chain acyl CoAs by the enzyme acyl CoA synthetase (ACSL). There are five known isoforms of ACSL (ACSL1,3,4,5,6) that vary in their tissue specificity and affinity for fatty acid substrates. To investigate the role of ACSL1 in the regulation of lipid metabolism, we used adenoviral-mediated gene transfer to overexpress ACSL1 in the human hepatoma cell-line HepG2 and in liver of rodents. Infection of HepG2 cells with the adenoviral construct AdACSL1 increased ACSL activity >10-fold compared with controls after 24 hours. HepG2 cells overexpressing ACSL1 had a 40% higher TG content (93±-3 compared to 67±-2 nmol/mg of protein in controls, p<0.05) after 24h exposure to 1mM oleate. Furthermore, ACSL1 overexpression produced a 60% increase in cellular LCACoA content (160±-6 compared to 100±-6 nmol/g of protein in controls, p<0.05), and increased ¹⁴C-oleate incorporation into TG without significantly altering fatty acid oxidation. In mice, AdACSL1 administration increased ACSL1 mRNA and protein more than 5-fold over controls at 4 days post infection. ACSL1 overexpression caused a 2-fold increase in TG content in mouse liver (39±4 compared to 20±2 µmicromol/g wet weight in controls, p<0.05) and overexpression in rat liver increased 1-¹⁴C palmitate clearance into liver triglyceride. These in vitro and in vivo results suggest a pivotal role for ACSL1 in regulating TG synthesis in liver.