A simple and accurate method was developed for the simultaneous determination of 13-cis- and all-trans-retinoic acid (13cRA, tRA), and retinol (ROH) in human serum using isocratic high-performance liquid chromatography (HPLC). The serum sample (0.2 ml) was diluted with 2 ml of acetonitrile-100 mM ammonium acetate (1:3, v/v), pH 5.5, and extracted with 5 ml of n-hexane. The extract was analyzed on an ODS column with a mobile phase consisting of 70 vols. of acetonitrile-methanol (2:1) and 30 vols. of 100 mM ammonium acetate, pH 7.0, at 50 degrees C. The retinoids were detected at 340 nm. Average recoveries were 88.4% for 13cRA, 82.5% for tRA at fortification levels of 5, 10 and 25 ng/ml, and 84.8% for ROH at 550 ng/ml. Within-day precision for normal human serum samples was 4.7% for 13cRA, 11.9% for tRA and 3.7% for ROH, and between-day precision was 10.4%, 14.2% and 4.7%, respectively. The limit of determination was 0.5 ng/ml in serum for the RAs. Mean concentration in 20 human sera was found to be 1.80 ng/ml for 13cRA, 1.77 ng/ml for tRA, and 487 ng/ml for ROH.