Enhancers increase the frequency of transcription initiation from linked promoter elements1, most probably as a result of the binding of specific proteins to the enhancer. The polyomavirus early region is expressed in differentiated mouse cells but not in undifferentiated embryonal carcinoma (EC) cells2,3. This host range is a function of the enhancer because polyomavirus mutants selected for growth in EC cells have mutations in the enhancer4–6 and the host range is reproduced in transfection assays using the mutant enhancers7. To understand the basis for this alteration in enhancer function, we have assayed extracts of EC cells for proteins that can interact with this sequence. We have detected a protein, present in a variety of cells, that can bind to the F441 mutant sequence, but binds only very poorly to the wild-type sequence. We conclude that this sequence alteration has probably generated a binding site for a positive-acting factor that allows the enhancer to function.