A sensitive and specific high-performance liquid chromatographic method was developed and validated for the determination of the third-generation retinoid (arotinoid) mofarotene (Ro 40-8757) in human, rat and dog plasma, using direct injection of deproteinated plasma samples, automated column switching (on-line solid-phase extraction) and ultraviolet detection. Plasma (0.5 ml) was deproteinated by adding ethanol (1 ml) containing the internal standard Ro 42-8659 (200 ng/ml). After centrifugation, 0.9 ml of the supernatant were directly injected onto a precolumn packed with C18 Corasil 37-50 microns. Polar plasma components were washed out from the precolumn using 1% ammonium acetate-acetic acid-acetonitrile (900:9:100, v/v/v). After valve switching, the pre-concentrated compounds were transferred to the analytical column (C18) in the backflush mode, separated by gradient elution and detected at 300 nm. The retention times (total run times) were approximately 15 and 20 min for the internal standard and mofarotene, respectively. The method was linear in the range 10-1000 ng/ml with a limit of quantification of 10 ng/ml. The mean recoveries were 80.4%, 81.7% and 77.8% (range 10-1000 ng/ml) and the inter-assay precision was 2.7% (range 20-1000 ng/ml), 1.5% and 2.0% (both range 100-1000 ng/ml) for human, rat and dog plasma, respectively. Mofarotene was found to be stable in human, rat and dog plasma stored at -20 degrees C for 3 months and 22 degrees C for 24 h. The method was successfully applied to clinical, pharmacokinetic and toxicokinetic studies.