The potential toxic effects of high extracellular concentrations of fatty acids were tested in β(INS-1)-cells cultured in the absence of serum, a condition known to alter cell survival in various systems. This may in part mimic the situation in type 1 or 2 diabetes where β-cells are already insulted by various stressful conditions, such as cytokines and oxidative stress. Serum removal caused, over a 36-h period, oxidative stress and an early impairment of mitochondrial function, as revealed by increased superoxide production and markedly reduced mitochondrial membrane potential, but a lack of cytochrome c and apoptosis-inducing factor release in the cytosol. The fatty acids palmitate and oleate considerably accelerated the apoptosis process in serum-starved cells, as revealed by fluorescence-activated cell sorting analysis, morphological changes, chromatin condensation, DNA laddering, poly(ADP-ribose) polymerase cleavage, cytochrome c and apoptosis-inducing factor release, and increased levels of Bax and cytosolic caspase-2. The fatty acids also increased nitric oxide production, apparently independently of inducible nitric oxide synthase induction. Under the same experimental conditions, elevated glucose alone had only a marginal effect on β-cell apoptosis. Together the results indicate that elevated concentrations of fatty acids are particularly efficient in accelerating the rate of apoptosis of already stressed β(INS-1)-cells displaying altered mitochondrial function, and that the mitochondrial arm of the apoptosis process is involved in β-cell lipotoxicity.