OBJECTIVE The principal commercial use of o-anisidine is believed to be as an intermediate in the manufacture of dyes. It has also been reported to be an intermediate in the manufacture of synthetic guaiacol and its derivatives. o-Anisidine is an urinary bladder carcinogen in mice and rats. The aim of the study was to investigate the kinetics of body distribution, excretion and biotransformation of o-anisidine in rats following a single, intraperitoneal administration. METHODS The tissue distribution and excretion of o-anisidine following i.p. administration of a single dose of 10 mg/kg was investigated using radiotracer [3H]. Metabolism of o-anisidine was investigated in the rats following i.p. administration of a single dose of 50 mg/kg using GC/MS technique. RESULTS After 72 h, about 72% of the given dose was excreted in urine. As indicated, urine proved to be the main route of tritium excretion. In all examined tissues, the highest concentrations of tritium were found 12 h after injection and the highest accumulation was detected in the liver, kidneys and in the muscle tissue. In urine, the following substances were identified and quantified by GC peak areas: N-acetyl-2-methoxyaniline and N-acetyl-4-hydroxy-2-methoxyaniline. CONCLUSIONS Prolonged tritium retention observed in the majority of tissues indicated that o-anisidine, especially in the case of repeated exposure, might accumulate in the body. The metabolism encompasses amine group acetylation and ring oxidation.