We have developed and validated a sensitive and selective assay for the quantification of paclitaxel and its metabolites 6 alpha, 3'-p-dihydroxypaclitaxel, 3'-p-hydroxypaclitaxel and 6 alpha-hydroxypaclitaxel in plasma, tissue, urine and faeces specimens of mice. Tissue and faeces were homogenized (approximately 0.1-0.2 g/ml) in bovine serum albumin (40 g/l) in water, and urine was diluted (1:5, v/v) in blank human plasma. Sample pretreatment involved liquid-liquid extraction of 200-1000 microliters of sample with diethyl ether followed by automated solid-phase extraction using cyano Bond Elut columns. 2'-Methylpaclitaxel was used as internal standard. The overall recovery of the sample pretreatment procedure ranged from 76 to 85%. In plasma, the lower limit of detection (LOD) and the lower limit of quantitation (LLQ) are 15 and 25 ng/ml, respectively, using 200 microliters of sample. In tissues, faeces and urine the LLQs are 25-100 ng/g, 125 ng/g and 25 ng/ml, respectively, using 1000 microliters (faeces: 200 microliters) of homogenized or diluted sample. The concentrations in the various biological matrices, for validation procedures spiked with known amounts of the test compounds, are read from calibration curves constructed in blank human plasma in the range 25-100,000 ng/ml for paclitaxel and 25-500 ng/ml for the metabolites. The accuracy and precision of the assay fall within the generally accepted criteria for bio-analytical assays.