Technical Abstract: Dietary micronutrient deficiencies are a major source of morbidity and mortality worldwide. Vitamin A deficiency is particularly devastating, causing blindness or corneal afflictions in 40 million children each year, and putting an additional 140-250 million at risk for related vitamin A deficiency disorders. A substantial number of these children subsist on a maize-based diet, particularly in sub-Saharan Africa. Maize varieties display tremendous natural variation for carotenoid composition and quantity in seed tissues including vitamin A precursors - beta-carotene and beta-cryptoxanthin. We used association analysis, linkage mapping, expression analysis, and mutagenesis to show that variation at the lycopene epsilon cyclase locus (lcyE) mediates the apportioning of carotenoids to the alpha-carotene and beta-carotene branches of this pathway, and thus is a key determinant of provitamin A content of the maize grain. Natural variation at four polymorphic sites explains nearly 60% of the variation and can produce a 5-fold or greater accumulation of beta versus alpha carotenoids, and a 3-fold increase in the proportion of provitamin A compounds. To facilitate breeding of provitamin A biofortified maize, PCR markers and germplasm stocks that allow for inexpensive screening and selection were developed. By selecting for specific lcyE alleles, breeders in developing countries can more efficiently and reliably produce maize grain with higher provitamin A levels.