No intensive study has been reported on the isolation and identification of the xanthophylls of freshly cut alfalfa. Zscheile et al. (7) mentioned briefly that they obtained lutein by chromatography of alfalfa leaves, and Strain (6) observed that egg yolks of chickens kept on a ration of wheat bran, milk, and fresh alfalfa contained minute traces of neoxanthin and flavoxanthin-like compounds, and considerable quantities of zeaxanthin, isolutein, and cryptoxanthin. Eventually it will be desirable to study the changes in the composition of the xanthophylls of alfalfa as growth of the plants progresses. This will require a knowledge of the pigments present and a method for determining them. The purpose of this paper is to present the results of a study on the chromatographic separation and subsequent identification of alfalfa xanthophylls. Extraction of Pigments Freshly harvested alfalfa (1 Kg.) was cut into one inch pieces and was comminuted with Skellysolve B and 95 per cent ethanol (1:3) in a Waring blendor. The pulp and solvent were separated by suction filtration with a buchner funnel, and the pulp was washed with a small amount of Skellysolve B. An equal volume of Skellysolve B was added to the extract and the mixture was allowed to stand in a separatory funnel until two layers formed. The alcohol layer was removed and was added to an equal volume of Skellysolve B. A volume of water corresponding to three times the combined volume of the alcohol and Skellysolve B was added to the mixture. The alcohol-water layer was discarded, and the two Skellysolve B extracts were combined and washed with water to remove traces