Technical Abstract: The presence of multiple enhancers and promoters within a single vector often provokes complicated mutual interaction and crosstalk, thereby, altering promoter specificity, which causes serious problems for precisely engineering gene function and agronomic traits in transgenic plants. Enhancer elements are known to be able to regulate their cognate promoters located either in proximity or at a distance. While there seems to be a general consensus concerning the ability of enhancers to exert their function by recruiting a selection of protein factors and delivering them to a competent promoter, the mechanism behind their function remains elusive. To gain a better understanding of these elements in a plant system, we analyzed the activation of a B-glucuronidase (GUS) reporter gene by the enhancer contained within the AGAMOUS second intron, both in the presence and absence of a minimal promoter. In addition, the long-distance activation function of the cauliflower mosaic virus 35S enhancer was examined by combining a cassette including the 35S enhancer to another comprising the AGI fused to the GUS coding region (with or without an intervening minimal promoter) in a head-to-head orientation. Our results indicate that both the AGI and 35S enhancers, which differ significantly in their species of origin and in the pattern of expression that they induce, appear to function in a similar manner to activate the expression of a nearby gene through the initiation of autonomous transcriptional events. Furthermore, the 35S enhancer was found to utilize a tracking and transcription mechanism to drive the activation of a distant target gene, providing further evidence for this animal- and yeast-derived model of long-distance enhancer action.