The present study was designed to define an experimental model of hepatocellular steatosis with a fat overaccumulation profile in which the metabolic and cytotoxic/apoptotic effects could be separated. This was accomplished by defining the experimental conditions of lipid exposure that lead to significant intracellular fat accumulation in the absence of overt cytotoxicity, therefore allowing to differentiate between cytotoxic and apoptotic effects. Palmitic (C16:0) and oleic (C18:1) acids are the most abundant fatty acids (FFAs) in liver triglycerides in both normal subjects and patients with nonalcoholic fatty liver disease (NAFLD). Therefore, human hepatocytes and HepG2 cells were incubated with a mixture of different proportions of saturated (palmitate) and unsaturated (oleate) FFAs to induce fat-overloading. Similar intracellular levels of lipid accumulation as in the human steatotic liver were achieved. Individual FFAs have a distinct inherent toxic potential. Fat accumulation, cytotoxicity and apoptosis in cells exposed to the FFA mixtures were investigated. The FFA mixture containing a low proportion of palmitic acid (oleate/palmitate, 2:1 ratio) is associated with minor toxic and apoptotic effects, thus representing a cellular model of steatosis that mimics benign chronic steatosis. On the other hand, a high proportion of palmitic acid (oleate/palmitate, 0:3 ratio) might represent a cellular model of steatosis in which saturated FFAs promote an acute harmful effect of fat overaccumulation in the liver. These hepatic cellular models are apparently suitable to experimentally investigate the impact of fat overaccumulation in the liver excluding other factors that could influence hepatocyte behaviour.