BACKGROUND: Cannabinoid receptors CB1 and CB2 are expressed in the liver, but their regulation in fatty hepatocytes is poorly documented. The aim of this study was to investigate the effects of selective CB1 or CB2 agonists on the expression of key regulators of lipid metabolism. METHODS: We used an in vitro model of fatty liver by treating immortalized human hepatocytes and HepG2 cells with oleic acid and the selective agonists arachidonyl-2-chloroethylamide (ACEA) (CB1, 12 nM) and (2-iodo-5-nitrophenyl)-[1-(1-methylpiperidin-2-ylmethyl)-1H-indol-3-yl]-methanone (AM1241) (CB2, 16 nM). The quantity of intracellular lipids was assessed using Oil-Red-O and a biochemical triglyceride assay. The expression of several proteins regulating endocannabinoid signalling and lipid metabolism was quantified by real-time polymerase chain reaction and by Western blot. RESULTS: Both CB1 and CB2 agonists dose-dependently increased the degree of steatosis of oleic acid-treated fatty hepatocytes. Cannabinoid receptors were downregulated in the presence of steatosis, and treatment with a CB2 agonist increased the expression of CB1. Carnitine palmitoyltransferase 1 was significantly overexpressed and sterol response element-binding protein (SREBP)-1c, fatty acid synthase and lecithin-cholesterol acetyltransferase (LCAT) were downregulated in fatty immortalized human hepatocytes. Treatment with the CB agonists ACEA and AM1241 partially reversed these changes, except for SREBP-1c. CB2, but not CB1, agonism decreased the expression of apolipoprotein B. In HepG2 cells, only LCAT resulted increased after treatment with CB agonists. CONCLUSIONS: Not only CB1 but also CB2 participated in the regulation of lipid metabolism in human-derived immortalized hepatocytes by regulating the expression of key enzymes of lipid synthesis and transport.