An HPLC method using paired-ion chromatography on RP C-18 material was developed. After sample clean up on XAD columns, mitoxantrone (Novantrone; dihydroxyanthracenedione) in concentrations below 1 ng/ml in serum and 0.2 ng/ml in urine were measurable with a coefficient of variation less than 9.3% at a wavelength of 658 nm. Four metabolites were separated in urine. The major metabolite cochromatographed with the synthesized dicarboxylic acid of mitoxantrone. Within 48 hours 4.4% of the administered dose was excreted in urine as mitoxantrone, 0.5% as metabolite 1 and 0.3% as metabolite 2. The pharmacokinetic parameters are adequately described by a three-compartment model with a terminal half-life of 214.8 hours, and a volume of distribution (ss) of 3792 litres. The total body clearance was 358 ml/min and the renal clearance was 26.2 ml/min.