The c-Myb protein is controlled by intramolecular interactions, and point mutations can enhance its oncogenic activity. We tested whether conformational changes regulate c-Myb and found that Cyp-40, a widely distributed cyclophilin and peptidyl-prolyl isomerase, could inhibit c-Myb DNA binding activity. Inhibition by Cyp-40 required both its C-terminal protein-interaction domain, which bound specifically to c-Myb, and its N-terminal catalytic domain and was blocked by the competitive inhibitor cyclosporin A. Cyp-40 failed to bind or inhibit the oncogenic derivative v-Myb, which has a mutated Cyp-40 binding site. These results suggest that mutations in v-Myb allow it to evade a negative regulatory mechanism mediated by enzymes such as Cyp-40, and implicate peptidyl-prolyl isomerases in the regulation of transcription, transformation, and differentiation.