An isocratic high-performance liquid chromatographic method specifically developed to allow simple and rapid determination of retinol concentrations in serum and plasma is reported. Retinol and retinol acetate (the internal standard) are extracted into butanol-ethyl acetate, with no subsequent evaporation step. Separation is achieved on a reversed-phase C-18 column, with a mobile phase consisting of acetonitrile-1% ammonium acetate (89:11), and UV detection at 313 nm. Recoveries of both retinol and the internal standard were 100%, and both compounds were stable in the extraction solvent for at least 2.5 h. Three anticoagulants (oxalate, citrate, EDTA) and perchloric acid (used in some methods to denature protein) all caused losses of retinol. Each run required 9 min; same-day coefficient of variation (C.V.) for identical samples averaged 2.5%; between-day C.V. was 6.4%; sensitivity was better than 10 ng/ml, while clinical concentrations were 400-1200 ng/ml. This method permits simple, rapid, sensitive, precise, and accurate determination of retinol using 0.5 ml serum or heparinized plasma.