Carotenoids are a diverse group of tetraterpenoid pigments found in plants, fungi, bacteria and some animals. They play vital roles in plants and provide important health benefits to mammals including humans. We previously reported the creation of a diverse population of transgenic maize plants expressing different carotenogenic gene combinations and exhibiting distinct metabolic phenotypes. Here we carried out an in depth targeted mRNA and metabolomic analysis of the pathway to characterize the specific impact of five carotenogenic transgenes and their interactions with twelve endogenous genes in four different transgenic lines representing distinct genotypes and phenotypes. We reconstructed the temporal profile of the carotenoid pathway during endosperm development at the mRNA and metabolic levels (for total and individual carotenoids) and investigated the impact of transgene expression on the endogenous pathway. These studies enabled us to investigate the extent of any interactions between the introduced transgenic and native partial carotenoid pathways during maize endosperm development. Importantly, we developed a theoretical model which explains these interactions, and suggest functioning modes for the pathway that identify intervention points allowing the maize endosperm carotenoid pathway to be engineered in a more effective and predictable manner. This article is protected by copyright. All rights reserved.