High performance liquid chromatography of yolks of hens fed a diet based on yellow corn, alfalfa, and soybeans revealed over 20 cartenoids. Lutein, lutein monester, lutein diester, 3'-oxolutein, cryptoxanthin, zeaxanthin, beta-carotene, and zeacarotene were identified by their retention times, visible absorption spectra, behavior on saponification, and their presence or absence when lutein was the primary carotenoid fed. Three weeks after placing the hens on a white corn-soy-based diet supplemented with lutein (20 micrograms/g diet), cryptoxanthin, zeaxanthin, and zeacarotene were undetectable in the yolk and lutein, lutein monoester, lutein diester, and 3'-oxolutein assumed new equilibrium concentrations. The data imply an esterification pathway and an oxidative pathway in laying hens for the metabolism of hydroxycarotenoids. Consideration of the concentrations and ratios of lutein and its metabolites in serum and yolk suggest a nonovarian site for the metabolism of lutein in laying hens.