SUMMARY The molecular foundations of evolution are difficult to trace because most protein sequences are virtually identical in closely related species. The largest fraction of sequence within the genome, however, is composed of noncoding sequences where regulatory elements locate to various sites. It has been suggested that changes in the activity of these elements may trigger evolutionary change. In Drosophila, the enhancer trap procedure identifies regulatory sequences in the genome after the insertion of a P-element–based construct. We generated new insertions and characterized their expression domains in the adult eye and larval imaginal disks using the white and LacZ reporter genes. Lines with robust expression patterns in D. melanogaster were analyzed in hybrids to test the conservation of regulatory mechanisms between species. Most of the enhancers used in this study modified their expression in hybrids with the mating species D. mauritiana and D. simulans. Expression changes resulted either in gain or loss of expression and were cell-type or hybrid-genome specific. Further characterization of a limited number of enhancers in D. melanogaster showed that expression domains could adapt to changes in cell number during development but not after the completion of cell proliferation. Also, expression of some enhancers appeared to be sensitive to heterochromatin from the Y but not the X chromosome. Taken together, these results demonstrate the high sensitivity of regulatory mechanisms of gene expression as a prime source of evolutionary change and suggest quantitative changes in available transcription factors as one of the mechanisms involved.