High-performance liquid chromatography (HPLC) was used to detect cis-trans isomers of a-and /3-carotene. Monitoring HPLC eluants at both 436 and 340 nm and calculating the peak height ratio, 340 nm/436 nm, permit detection of isomers in carotene-petroleum ether solutions and plant extracts. Separation of individual isomers was achieved on thin-layer chromatography (TLC) plates prepared with calcium hydroxide layers. Equilibration of the plates to 44% relative humidity prior to development with 1.5% p-methylanisole in petroleum ether was necessary to achieve optimal resolution. No isomeric carotenoids were found in the fresh yellow-orange vegetables, but analyses of chlorophyll-containing vegetables indicated that isomers were present. Heat-processed products contained a variety of cis isomers. Sample handling, saponification methods, and sample pH did not influence the formation of isomers.