Long chain acyl-CoA synthetases (ACSL) activate fatty acids (FA) and provide substrates for both anabolic and catabolic pathways. We have hypothesized that each of the five ACSL isoforms partitions FA toward specific downstream pathways. Acsl1 mRNA is increased in cells under both lipogenic and oxidative conditions. To elucidate the role of ACSL1 in hepatic lipid metabolism, we overexpressed an Acsl1 adenovirus construct (Ad-Acsl1) in rat primary hepatocytes. Ad-ACSL1, located on the endoplasmic reticulum but not on mitochondria or plasma membrane, increased ACS specific activity 3.7-fold. With 100 or 750 μM [1-14C]oleate, Ad-Acsl1 increased oleate incorporation into diacylglycerol and phospholipids, particularly phosphatidylethanolamine and phosphatidylinositol, and decreased incorporation into cholesterol esters and secreted triacylglycerol. Ad-Acsl1 did not alter oleate incorporation into triacylglycerol, β-oxidation products, or total amount of FA metabolized. In pulse-chase experiments to examine the effects of Ad-Acsl1 on lipid turnover, more labeled triacylglycerol and phospholipid, but less labeled diacylglycerol, remained in Ad-Acsl1 cells, suggesting that ACSL1 increased reacylation of hydrolyzed oleate derived from triacylglycerol and diacylglycerol. In addition, less hydrolyzed oleate was used for cholesterol ester synthesis and β-oxidation. The increase in [1,2,3- 3H]glycerol incorporation into diacylglycerol and phospholipid was similar to the increase with [14C]oleate labeling suggesting that ACSL1 increased de novo synthesis. Labeling Ad-Acsl1 cells with [ 14C]acetate increased triacylglycerol synthesis but did not channel endogenous FA away from cholesterol ester synthesis. Thus, consistent with the hypothesis that individual ACSLs partition FA, Ad-Acsl1 increased FA reacylation and channeled FA toward diacylglycerol and phospholipid synthesis and away from cholesterol ester synthesis.