Technical Abstract: The carpel- and stamen-specific AtAGIP promoter derived from the Arabidopsis AGAMOUS (AG) second intron/enhancer is ideal for engineering complete sterility, but it is highly host-specific. To ascertain that a chimeric promoter with similar tissue specificity can be created for species other than Arabidopsis, we isolated two similar but distinct AG second intron/enhancers from tobacco (NtAGI-1 and NtAGI-2) and analyzed their ability to drive floral organ-specific expression in plants through the creation of forward- and reverse-oriented chimeric promoters, fNtAGIP1, rNtAGIP1, fNtAGIP2 and rNtAGIP2. Transgenic analyses confirmed that all four promoters, like the AtAGIP, drive carpel- and stamen-specific expression without any leaky activity in vegetative tissues as demonstrated by tissue-specific GUS expression and cytotoxic gene (DT-A)-mediated tissue ablation. Unlike the AtAGIP, both tobacco enhancers displayed additional weak petal- and sepal-specific activity as revealed by partially retarded petals and sepals in ablated flowers. Unexpectedly, the activity of the promoters is influenced by the orientation of the incorporated enhancers with reverse-oriented enhancers exhibiting approximately double the effectiveness of forward-oriented enhancers; a phenomenon which has not been observed for the AtAGIP promoter. Our results suggest that the tissue-specificity of the tobacco AGI enhancers is evolutionarily conserved and has not diverged between the two enhancers, as is the case in rice and maize. Furthermore, we show that the NtAGI-derived promoters are effective for engineering complete sterility and that the resulting trait is mitotically stable, which is critical for the long-term containment of seed-, pollen- and fruit-mediated gene flow in field conditions.