We describe a specific assay for serum provitamin A (alpha- and beta-carotene) by high pressure liquid chromatography (HPLC). The system separates alpha- and beta-carotene in 7.4 min using a C18 muBondapak, 10-micron particle size column with a mobile phase of acetonitrile-chloroform 92:8 at 2 ml per min and a 462 nm detector. The HPLC assay had a recovery of 94.8% of added beta-carotene and, at a serum concentration of 215.2 micrograms/L, had within-run and between-run precisions of 3.1% and 3.6%, respectively. In 65 subjects, the HPLC-determined provitamin A (alpha- and beta-carotene) value was 343 +/- 166 micrograms/L and averaged 23.4 +/- 7.9% (range 9-43%) of the values obtained by a traditional colorimetric assay for total serum "carotenes." Although total serum carotenes showed no relationship to serum vitamin A (r = -0.048; P = 0.78), HPLC-determined alpha- and beta-carotene was significantly inversely correlated (r = -0.357; P = 0.05).