Computational methods can be used to identify DNA sequence motifs that have been conserved through evolution, as well as motifs that correspond to recognition sites for known DNA-binding proteins. These computational methods, when combined with chromatin immunoprecipitation and other basic experiments, can provide preliminary insight into the elements and factors that regulate a gene of interest. When pursuing a more complete understanding of a control region of interest, a comprehensive mutant analysis should generally be performed as a critical step toward more advanced functional studies. This article describes strategies for such a comprehensive analysis. It also summarizes the insights provided by a comprehensive mutant analysis versus a phylogenetic analysis.