The present study was undertaken to quantitate the major carotenoids in human milk, monitoring their variance among fore, mid, and hind mature milk samples; during morning, midday, and evening, and among different weeks of lactation. Twenty-three mothers, 6 weeks to 16 weeks postpartum, participated in the study. Reversed-phase high performance liquid chromatography (RP-HPLC) was used to identify and quantitate the major carotenoids found in human milk, including lutein/zeaxanthin, beta cryptoxanthin, lycopene, alpha carotene, and beta carotene. To determine the validity of analyzing carotenoids in frozen milk, freshly expressed and subsequently frozen milk samples were analyzed for four mothers. Freezing did not alter carotenoid concentrations. Wide variations in carotenoid concentrations existed within and between the mothers. Major carotenoid totals ranged from 29 nM to 493 nM. Complete breast expressions at three periods within a day and at specified weekly intervals allowed the accurate determination of diurnal as well as longitudinal carotenoid concentrations. Diurnal evaluation of carotenoid concentrations suggested a peak at midday and highest longitudinal concentrations were at 10 to 12 weeks. Neither of these trends was statistically significant. The analysis of fore, mid, and hind milk carotenoid concentrations demonstrated that hind milk was significantly higher than fore or mid milk ( P < 0.05).