The RFX DNA binding domain is a novel motif that has been conserved in a growing number of dimeric DNA-binding proteins, having diverse regulatory functions, in eukaryotic organisms ranging from yeasts to humans. To characterize this novel motif, we have performed a detailed dissection of the site-specific DNA binding activity of RFX1, a prototypical member of the RFX family. First, we have performed a site selection procedure to define the consensus binding site of RFX1. Second, we have developed a new mutagenesis-selection procedure to derive a precise consensus motif, and to test the accuracy of a secondary structure prediction, for the RFX domain. Third, a modification of this procedure has allowed us to isolate altered-specificity RFX1 mutants. These results should facilitate the identification both of additional candidate genes controlled by RFX1 and of new members of the RFX family. Moreover, the altered-specificity RFX1 mutants represent valuable tools that will permit the function of RFX1 to be analyzed in vivo without interference from the ubiquitously expressed endogenous protein. Finally, the simplicity, efficiency, and versatility of the selection procedure we have developed make it of general value for the determination of consensus motifs, and for the isolation of mutants exhibiting altered functional properties, for large protein domains involved in protein-DNA as well as protein-protein interactions.