Structural and functional constraints are known to play a major role in restricting the path of evolution of protein activities. However, constraints acting on evolving transcriptional regulatory sequences, e.g. enhancers, are largely unknown. Recently, we elucidated how a novel expression pattern of the Neprilysin-1 ( Nep1 ) gene in the optic lobe of Drosophila santomea evolved via co-option of existing enhancer activities. Drosophila santomea , which has diverged from Drosophila yakuba by approximately 400 000 years has accumulated four fixed mutations that each contribute to the full activity of this enhancer. Recreating and testing the optic lobe enhancer of the ancestor of D. santomea and D. yakuba revealed that the strong D. santomea enhancer activity evolved from a weak ancestral activity. Because each mutation on the path from the D. yakuba/santomea ancestor to modern-day D. santomea contributes to the newly derived optic lobe enhancer activity, we sought here to use this system to study the path of evolution of enhancer sequences. We inferred likely paths of evolution of this enhancer by observing the transcriptional output of all possible intermediate steps between the ancestral D. yakuba/santomea enhancer and the modern D. santomea enhancer. Many possible paths had epistatic and cooperative effects. Furthermore, we found that several paths significantly increased ectopic transcriptional activity or affected existing enhancer activities from which the novel activity was co-opted. We suggest that these attributes highlight constraints that guide the path of evolution of enhancers.