The xanthophyll carotenoids lutein (L), zeaxanthin (Z) and meso-zeaxanthin (MZ) are found at the macula, the central part of the retina, where they are referred to as macular pigment (MP). MP is studied in human subjects because of its proven role in enhancing visual function and its putative role in protecting against age-related macular degeneration. These benefits are probably due to the antioxidant and short-wavelength filtering properties of MP. It is known that eggs are a dietary source of L and Z. This experiment was designed to measure the egg yolk carotenoid response to hen supplementation with L, Z and MZ. A total of forty hens were used in the trial and were divided into eight groups of five hens. Each group was supplemented (with about 140 mg active xanthophylls/kg feed) with one of the following oil-based carotenoid formulations for 6 weeks: unesterified L (group 1); L diacetate (group 2); unesterified Z (group 3); Z diacetate (group 4); unesterified MZ (group 5); MZ diacetate (group 6); L–MZ (1:1) diacetate mixture (group 7); L–MZ diacetate (1:3) mixture (group 8). Yolk carotenoid content was analysed weekly (in four randomly selected eggs) by HPLC. We found that hens supplemented with Z diacetate and MZ diacetate produced eggs with significantly greater carotenoid concentrations than their free form counterparts. This finding potentially represents the development of a novel food, suitable to increase MP and its constituent carotenoids in serum.