The simian virus 40 72-base-pair repeats substituted for the polyomavirus enhancer, allowing replication and transcription in mouse 3T6 but not monkey CV-1 cells. A polyomavirus genome containing the entire simian virus 40 control region replicated at low levels in 3T6 and CV-1 cells; however, transcripts were detected only in 3T6 cells. Our results suggest that the simian virus 40 72-base-pair repeats are unable to alter the host species specificity of the complete polyomavirus genome.