Allosteric DNA oligonucleotides are potentially useful diagnostic reagents. Here we develop a model system for the study of allosteric interactions in DNAs. A DNA that binds either Cibacron blue or cholic acid was isolated and partially characterized. Isolation was performed using a multi-stage SELEX. First, short oligos that bind either Cibacron blue or cholic acid were enriched from random oligonucleotide pools. Then, members of the two pools were fused to form longer oligos, which were then selected for theability to bind Cibacron blue columns and elute with cholic acid. One resulting isolate (A22) was studied. Dye- and cholate-binding functions can be separated on sequences from the 5'- and 3'-regions, respectively. Ligand-column affinity assays indicate that each domain binds only its respective ligand. However, the full-length A22 will bind either dye or cholate columns and elute with the other ligand, as if binding by the ligands is mutually exclusive. Furthermore, S1 nuclease protection assays show that Cibacron blue causes a structural change in A22 and that cholic acid inhibits this change. This system will be useful for elucidating mechanisms of allosteric interactions in synthetic DNAs.