||Pristanic acid and phytanic acid are branched-chain fatty acids, which play an important role in diseases with peroxisomal impairment, like Refsum disease (MIM 266500), Zellwegers syndrome and alpha-methylacyl-CoA racemase deficiency (MIM 604489). Several studies revealed that the toxic activity of phytanic acid is mediated by multiple mitochondrial dysfunctions. However, the action of pristanic acid on brain cells is still completely unknown. Here, we exposed astrocytes, oligodendrocytes and neurons in mixed culture to pristanic acid and phytanic acid to analyse cellular consequences. Pristanic acid exerts a strong cytotoxic activity on brain cells, displayed by dramatic Ca2+ deregulation, in situ mitochondrial depolarization and cell death. Interestingly, pristanic acid strongly induced generation of reactive oxygen species (ROS), whereas phytanic acid exerts weaker effects on ROS production. In conclusion, pristanic acid as well as phytanic acid induced a complex array of toxic activities with mitochondrial dysfunction and Ca2+ deregulation.