A specific and sensitive capillary gas chromatography-mass spectrometry assay for the determination of tolterodine and the 5-hydroxymethyl metabolite (Labcode DD 01) in plasma, serum and urine is described. Extraction of the analytes was performed with liquid/liquid or solid-phase extraction prior to derivatisation with a silyl reagent. The derivatives were quantified by selected ion monitoring mass spectrometry using deuterium-labelled internal standards. A single level calibration curve was utilised for quantification of plasma, serum and urine concentrations of tolterodine and DD 01. The accuracy (inter- and intra-day) for both analytes was within 87-110% in the range 0.5 and 50 ng ml-1 and precision was better than 90%. Overall, this method was shown to be reliable for pharmacokinetic assays of tolterodine and the metabolite DD 01 in samples from preclinical and clinical studies.