A reverse phase liquid chromatographic (LC) system has been developed for separating the main naturally occurring carotenoids that have provitamin A activity. The system produces baseline separation of beta-carotene, alpha-carotene, and beta-cryptoxanthin (beta, beta-carotene-3-ol, 472-70-8) from biologically inactive zeinoxanthin (beta, epsilon-carotene-3-ol, 24480-38-4) and from a pigment believed to be alpha-cryptoxanthin (beta, epsilon-carotene-3'-ol). Some cis-isomers are also separated. These separations are obtained on a C-18 column, isocratically, with methanol-chloroform eluant. For quantitation, peak areas from detection at 475 nm are compared with that of an internal standard, 1-(phenylazo)-2-naphthalenol (842-07-9), which elutes prior to the provitamins. Provitamin amounts are calculated from absorbance ratios. Prior to LC, esters are saponified, and interfering pigments are removed from ester-free extracts by adsorption on magnesia.