In the mature retina, the components of the macular pigment, lutein (L), R,R-zeaxanthin (RRZ), R,S-zeaxanthin (RSZ, meso-zeaxanthin) are most concentrated in the central macula. L and RRZ are of dietary origin but RSZ is produced in situ from L. The relative proportions of L and Z isomers vary across the retina with eccentricity in the adult retina. Early reports have shown that during development, the proportions of L and Z isomers undergo changes as the total pigment levels increase. The methods described here demonstrate the unique utility of chiral phase HPLC to measure the amounts of L, RRZ, and RSZ, discriminating between the two zeaxanthin stereoisomers. In three concentric retinal sections of macaque retinas chiral phase HPLC has been employed to document the developmental changes in the distribution of each L, RSZ, and RRZ during the period just prior to full term gestation through 19 months after birth. The net rate of accumulation of carotenoids within the central retina during the first 20 months is quasi-linear and fit by a linear regression. During development, the rate of transport of L (0.12 (± 0.033) ng mm− 2 mo− 1 (SE)) into the central 2 mm of the retina is double that of RRZ (0.062 (± 0.02) ng mm− 2 mo− 1 (SE)). The rate of accumulation of RSZ (0.06 (± 0.01) ng mm− 2 mo− 1 (SE)) is comparable to that of RRZ. In the peripheral retina, the rates of accumulation of L and RRZ are not correlated with increasing age, whereas accumulation of RSZ does correlate with age. The changing proportions of L to Z isomers in the central retina during development are explained by the rates for carotenoid accumulation within the central retina. At birth, the macular pigment in the central retina is dominated by L and RRZ, 0.35 ± 0.11 and 0.21 ± 0.054 ng mm− 2. In the central retina, RSZ was rarely detected in the youngest tissues analyzed. It can be estimated to represent 6% of the total macular pigment (0.033 ± 0.11 ng mm− 2) at birth based on extrapolation from measurements in the peripheral retina and the ratio of L/(RRZ + RSZ) is ≈ 1.5. At maturity, the concentrations for L, RRZ, and RSZ in the central macaque retina are estimated to be 1.7, 1.8 and 1.08 ng mm− 2, with L/(RRZ + RSZ) being 0.6.