Reverse-phase high-performance liquid chromatography was used for the simultaneous determination of retinol, alpha-tocopherol, lycopene, alpha-carotene, and beta-carotene in human plasma. A multiple solvent system of methanol followed by a mixture of methanol:acetonitrile:chloroform (47:42:11) provided clear separation of these compounds. With retinyl acetate as an internal standard, standard curves were developed for each compound on the basis of peak area ratios. The coefficient of variation was less than 10% in all cases within a run. Between-run reproducibility was within +/- 2 standard deviations. The method required 100 to 200 microliter of plasma and 16 min of elution time per sample. Stability studies showed plasma samples could be stored at -10 degrees C with occasional freeze-thaw cycles for 3 to 5 weeks. This method should prove useful in clinical and epidemiological work.