A fully automated gradient high-performance liquid chromatographic method for the determination of isotretinoin, tretinoin and their 4-oxo metabolites in plasma was developed, using the column-switching technique. After dilution with an internal standard solution containing 20% acetonitrile, 0.5 ml of the sample was injected onto a precolumn (17 X 4.6 mm I.D.), filled with C18 Corasil 37-53 micron. Proteins and polar plasma components were washed out using 1% ammonium acetate-acetonitrile (9:1, v/v) as mobile phase 1. After valve switching, the retained components were transferred to the analytical column in the backflush mode, separated by gradient elution and detected at 360 nm by UV detection. Using two coupled reversed-phase columns (125 mm long), the separation of cis and trans isomers was possible, and all four compounds could be quantified down to 2 ng/ml of plasma. The inter-assay precision in the concentration range 20-100 ng/ml was between 1.0 and 4.7% for all compounds.