Alcoholic steatohepatitis (ASH) and nonalcoholic steatohepatitis (NASH) are the most frequent conditions leading to elevated liver enzymes and liver cirrhosis, respectively, in the Western world. However, despite strong epidemiological evidence for combined effects of alcohol consumption and hepatic lipid accumulation on the progression of liver injury, the mutual interaction of the pathophysiological mechanisms is incompletely understood. The aim of this study was to establish an in vitro model for joint effects of alcohol and lipids on hepatic steatosis and inflammation. Herein, we particularly wanted to assess the role of CYP2E1, which activates several hepatotoxins and contributes to alcoholic liver damage and the role of autophagy, which has been emerged as new mechanism for alcohol-induced liver injury. Initially, we established the dose range in which neither alcohol nor incubation with the free fatty acid (FFA) oleate affected viability or mitochondrial activity in primary human hepatocytes (PHH) and HepG2 hepatoma cells. Subsequently, we assessed the combined effect of alcohol (50 mM) and oleate (0.2 mM) on hepatocellular lipid accumulation and inflammation in PHH. Under these conditions, alcohol significantly enhanced oleate induced cellular triglyceride content, while alcohol alone had only a minimal effect on hepatocellular lipid content. Analysis of heme oxygenase-1 (HMOX-1) expression and malondialdehyde levels (MDA) revealed that the combination of alcohol and oleate caused significantly higher oxidative stress and lipid peroxidation than either of the two substances alone. The CYP2E1 inhibitor chlormethiazole and the antioxidant N-acetylcysteine blunted these combined effects of alcohol and oleate on hepatocellular lipid accumulation and inflammation in PHH. In contrast to HepG2 C34 cells which do not express CYP2E1, HepG2 E47 cells which express CYP2E1 exhibited similar joint effects of alcohol and oleate as observed in PHH. The combination of alcohol and FFA induced also significantly autophagy markers more than the each stimulus alone. Inhibition or induction of autophagy led to exacerbate or abrogate the combined effects of alcohol and FFA on lipid accumulation, lipid peroxidation and inflammation, respectively. In summary, our new model allows the investigation of isolated or joint effects of alcohol and FFA on hepatocellular lipid metabolisms and inflammatory signaling. Our present findings indicate that CYP2E1, CYP2E1-derived reactive oxygen species (ROS) and autophagy play a crucial role in mediating the synergistic effects of alcohol and lipids on hepatic steatosis, oxidative stress and inflammation.