The even-skipped (eve) promoter contains a series of enhancers that control the expression of different segmentation stripes in the Drosophila embryo. The stripe 3 enhancer is located 1.7 kb upstream of the stripe 2 enhancer. Here we demonstrate that these enhancers must be physically separated by a minimum distance for proper stripe expression. When they are directly coupled in either orientation, the enhancers generate abnormal patterns of expression in the early embryo. For example, the levels of stripe 2 expression are augmented and there is a posterior expansion of the pattern when the stripe 3 enhancer is positioned immediately upstream of the stripe 2 enhancer. Despite this spacing requirement, the order of the enhancers within the eve promoter can be reversed without affecting the normal expression pattern. These results suggest that spacing maintains the autonomous activities of the stripe enhancers and that interactions between enhancers can generate novel patterns of gene expression.