The study aimed to directly measure in vivo superoxide radical (O2−) a direct indicator of oxidative stress, in the brain of rats with experimentally induced obstructive jaundice by employing a new quantitative ultrasensitive fluorescent assay requiring minimum sample. O2− anion is specific for dihydroethidine (DHE) and upon reaction gives a characteristic product, namely 2-OH-ethidium. Ten male rats underwent laparotomy and were divided into two groups: I, sham operated and II bile duct ligation. Ten days later, following injection with DHE (a O2− trap), all animals were killed and samples from cerebral cortex, midbrain and cerebellum were removed for analysis. It was shown that compared to group I, in group II the O2− was increased by 67% in the cerebral cortex and by 37% in the midbrain as a consequence of experimental obstructive jaundice, while its levels were unaffected in the cerebellum. The data in this experimental obstructive jaundice model imply a region-specific increase of O2− formation rate, being higher in cerebral cortex, less so in the midbrain and not at all in cerebellum.