Lutein and zeaxanthin are xanthophylls that can be found highly concentrated in the macula of the retina. They are thought to protect the macula through their role as blue-light filters and because of their antioxidant and singlet oxygen quenching properties. Examination of metabolites unique to lutein and zeaxanthin such as 3'-dehydro-lutein, and of their stereochemistry may provide insight to the mechanism by which they are formed and by which they exert protection. To evaluate the formation of such metabolites, eleven monkeys were raised on a xanthophyll-free diet, and supplemented with pure lutein or pure zeaxanthin (2.2 mg/kg body weight/d). The period of supplementation ranged between 12 and 92 weeks. At study start and throughout the study, serum samples were taken and analyzed for xanthophylls using different HPLC systems. Xanthophyll metabolites were identified using UV/VIS and HR-MS detection. Lutein and zeaxanthin metabolites were found in detectable amounts with 3'-dehydro-lutein being a common metabolite of both. Using chiral-phase HPLC, two diastereomers, (3R,6'R)-3'-dehydro-lutein and (3R,6'S)-3'-dehydro-lutein, were identified and shown to be present in nearly equimolar amounts. A pathway for their formation from either lutein or zeaxanthin is proposed. These findings were comparable to results obtained with human plasma.