Quantitative enzyme-linked immunosorbent assays were used to characterize the geographical (locations) and temporal (through 6 wk) expression of CrylAc, from Bacillus thuringiensis variety kurstaki, and Cry1F, from B. thuringiensis variety aizawai, in transgenic cotton, Gossypium hirsutum L., plant structures. Terminal leaves, squares (flower buds), flowers, bolls (fruit), and mature leaves located five and eight nodes below the terminal apex were sampled during weeks 2, 4, and 6 after the initiation of anthesis. The effect of location (environment) significantly influenced protein expression levels, although similar trends were observed across locations. Cry1F was expressed at levels greater (1.1-29.0-fold) than that for CrylAc in all structures with exception to flowers. In contrast, the level of CrylAc in flowers was generally greater than Cry1F. Within each sampling period, concentrations of Cry1F in mature leaves (five and eight node) were greater than that for other structures. Expression was also greater for older, eight-node mature leaves than younger, five-node mature leaves. CrylAc expression in bolls was lowest compared with terminal leaves, squares, flowers, and mature leaves, which expressed at similar concentrations. Cry1F expression increased through time for mature leaves and terminal leaves; whereas, a decline in Cry1Ac protein concentration was observed for terminal leaves and bolls. The data presented here provides a means to understand observed levels of efficacy (patterns of insect damage) by comparing the spatial and temporal dynamics of expression for Cry1Ac and Cry1F in PhytoGen 440W transgenic cotton.