A high-performance liquid chromatographic method with automated column switching was developed for the simultaneous determination of 13-cis-3-hydroxyretinoic acid, all-trans-3-hydroxyretinoic acid and their metabolites 13-cis-3-hydroxy-4-oxo-retinoic acid and all-trans-3-hydroxy-4-oxo-retinoic acid in plasma samples from man, rat, dog, rabbit and mouse. The method consists of deproteination of plasma (0.4 ml) with ethanol (1.5 ml), containing the internal standard Ro 12-7310. After centrifugation, 1.4 ml of the supernatant was directly injected onto the precolumn (PC) (4 x 4 mm) packed with LiChrospher 100 RP-18 (5 microm). Ammonium acetate (0.02%)-acetic acid-ethanol (100:3:4, v/v/v) was used as mobile phase M1A during injection, as well as to decrease the elution strength of the injection solution by on-line addition using a T-piece (M1B). After valve switching, the retained components were transferred to the analytical column (AC), separated by gradient elution and detected at 360 nm. Two coupled Purospher 100 RP-18 endcapped columns (both 250 x 4 mm) were used for the separation, together with a mobile phase consisting of acetonitrile-water-10% ammonium acetate-acetic acid, (A), 540:450:2:30 (v/v/v/v), (B), 600:350:2:30 (v/v/v/v), and (C), 950:40:2:30 (v/v/v/v). The method was linear in the range 1-500 ng ml(-1), at least, with a quantification limit of 1 ng ml(-1). The mean recoveries from human plasma were 100-107% and the mean inter-assay precision was 2.0-4.7% (range 1-500 ng ml(-1)). Similar results were obtained for animal plasma. The analytes were stable in the plasma of all investigated species stored at -20 degrees C for 3 months, at least. The method was successfully applied to clinical and toxicokinetic studies.