There is increasing evidence showing the involvement of unsaturated free fatty acids in cell death pathways, particularly in the context of apoptotic signalling. Our previous in vitro study has demonstrated that oleic acid, a monounsaturated fatty acid, reduces phosphorylation of proapoptotic Bad through activation of protein phosphatase type 2Cbeta. In the present study, we attempted to investigate the role of oleic acid in neuronal apoptosis using different types of cell cultures, and, furthermore, to explore the underlying mechanism with regard to its effect on Bad expression. As revealed by nuclear staining, oleic acid caused a concentration- and time-dependent damage with typical apoptotic features in cortical and hippocampal cultures from embryonic and neonatal rats, respectively, as well as in human neuroblastoma SH-SY5Y cells. In mixed hippocampal cultures, nearly all neurons were damaged at 24 h after the treatment, while damage of astrocytes was detected 48 h after adding this fatty acid, suggesting that neurons were more vulnerable than astrocytes. Nile blue staining showed that oleic acid and oleic acid methyl ester were both taken up by the neurons within 30 min. In contrast to oleic acid, oleic acid methyl ester did not change cell viability demonstrating that oleic acid-induced cell death was not due to an overload of the cells with lipids. Caspase-3 activity was not increased by oleic acid in cultured hippocampal cells. Western blot analysis of phospho-Ser112 Bad and the total Bad in cultured hippocampal cells revealed a significant decrease in the ratio of phospho-Ser112 Bad to total Bad in a time- and concentration-dependent manner after the exposure with oleic acid. We conclude that oleic acid induces neuronal apoptosis through a caspase-3-independent mechanism involving dephosphorylation of Bad.