Dexamethasone (DXM) is a synthetic glucocorticoid that is authorized for therapeutic use in veterinary medicine. The European Community (EC) fixed a maximum residue limit (MRL) at 2ng/g for liver, 0.75ng/g for muscle and kidney tissues, and 0.3ng/ml for milk, while its use as growth-promoter is completely banned. The purpose of this study was to develop and validate a simple and reliable method to determine DXM residues in bovine milk. Milk proteins were removed by the addition of concentrated trichloroacetic acid and paper filtration. Solid-phase extraction clean-up on a C18 reversed phase column was performed to obtain an extract suitable for liquid chromatography-tandem mass spectrometry (LC-MS/MS) analysis. Chromatographic separation of DXM and the internal standard desoximetasone, was achieved on a PLRP-S polymeric reversed phase column, using a mixture of 0.1% (v/v) acetic acid in water (mobile phase A) and acetonitrile (mobile phase B) as the mobile phases. They were identified using the MS/MS detection technique, and were subsequently quantified. The method has been validated according to the requirements of the EC at 0.15, 0.30 and 0.60ng/ml (being half the MRL, the MRL and double the MRL levels fixed by the EC). Calibration graphs were prepared in the 0.15-5ng/ml range and good linearity was achieved (r>or=0.99 and goodness of fit <or=10%). A limit of quantification of 0.15ng/ml, i.e. half the MRL, was obtained. The limit of detection was 41pg/ml. The decision limit (CCalpha) and detection capability (CCbeta) were 0.48 and 0.76ng/ml, respectively. The within-day and between-day precisions, expressed as R.S.D. values, were all below the maximum allowed R.S.D. values calculated according to the Horwitz equation. The results for accuracy fell within the -50 to +20% range. Recovery was 56%. The method was used for the quantitative determination of DXM residues in milk after intravenous administration of DXM to lactating cows to determine its depletion kinetics.