Iron deficiency is an important public health problem that is estimated to affect over one-half the world population. Improving the nutritional quality of staple food crops such as maize, by developing varieties with high bioavailable iron represents a sustainable and cost effective approach to alleviating iron malnutrition. The aim of our study was to use a Caco-2 cell / quantitative trait loci (QTL) / and poultry approach to discover the genes that influence maize grain iron concentration and amount of bioavailable iron. After several breeding cycles guided by measurements of iron bioavailability with the Caco-2 cell model, we identified two maize lines with identical grain iron concentration (20 [mu]g/g), but one line contained double the amount of bioavailable iron (p [LESS THAN] 0.001). Three QTL were found to influence the bioavailability of iron in the two maize lines. Measurement of iron status in a poultry model confirmed the results of the Caco-2 cell model. Some of the lines screened using the Caco-2 cell / QTL / and poultry approach have been grown at multiple locations and data show a strong genotype x environment (GxE) interaction. We conclude that conventional breeding can significantly improve iron bioavailability in maize grain and that the GxE effect plays a major role in iron bioavailability. Human feeding trials should be conducted to determine the efficacy of consuming the high bioavailable iron maize.