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Gene transcription can be regulated by promoter competition when multiple promoters are available for a single enhancer. The converse, however, is largely undescribed. Here we report an occurrence of several enhancers competing for one promoter (enhancer interference) and propose its underlying mechanism. The promoter targeting sequence from the AbdominaI-B locus of the Drosophila bithorax complex overcomes the enhancer-blocking activity of insulators in transgenic embryos. It may facilitate the long-range enhancer-promoter communication in Abdominal-B by bypassing insulator elements such as Frontabdominal-7 and Front-abdominal-8. In transgenic embryos, the anti-insulator activity allowed both an insulator-blocked enhancer and a nonblocked enhancer to contact the same promoter. We found that these two enhancers exhibited mutual inhibition or mutual exclusion in cells where both are transcriptionally active. This enhancer interference occurred at the level of enhancer-promoter communication. It occurred only between enhancers on opposite sides of an insulator and depended on enhancer activity. We hypothesize that enhancer interference limits the interaction of the AbdominaI-B promoter to the enhancer(s) from only one regulatory domain in a specific abdominal segment. Abd-B | Fab-8 | IAB8 | NEE