The degree of damage to DNA following ifosfamide (IFO) treatment may be linked to the therapeutic efficacy. The pharmacokinetics and metabolism of IFO were studied in 19 paediatric patients, mostly with rhabdomyosarcoma or Ewings sarcoma. Ifosfamide was dosed either as a continuous infusion or as fractionated doses over 2 or 3 days. Samples of peripheral blood lymphocytes were obtained during and up to 96 h after treatment, and again prior to the next cycle of chemotherapy. DNA damage was measured using the alkaline COMET assay, and quantified as the percentage of highly damaged cells per sample. Samples were also taken for the determination of IFO and metabolites. Pharmacokinetics and metabolism of IFO were comparable with previous studies. Elevations in DNA damage could be determined in all patients after IFO administration. The degree of damage increased to a peak at 72 h, but had returned to pretreatment values prior to the next dose of chemotherapy. There was a good correlation between area under the curve of IFO and the cumulative percentage of cells with DNA damage (r2 = 0.554, P = 0.004), but only in those patients receiving fractionated dosing. The latter patients had more DNA damage (mean ± s.d., 2736 ± 597) than those patients in whom IFO was administered by continuous infusion (1453 ± 730). The COMET assay can be used to quantify DNA damage following IFO therapy. Fractionated dosing causes a greater degree of DNA damage, which may suggest a greater degree of efficacy, with a good correlation between pharmacokinetic and pharmacodynamic data.