The gene coding for the human cytomegalovirus major immediate-early 72-kilodalton protein was cloned into simian virus origin of DNA replication plasmid pSVOd. Transfection of this plasmid (pSVCC2) into cells constitutively expressing the simian virus 40 T-antigen resulted in readily detectable levels of immediate-early region 1-specific RNA and protein. Partial restriction enzyme digestion of pSVCC2 was used to generate specific amino acid deletions within the 72-kilodalton protein. Mutant delta S12, which contained a deletion of 145 amino acids at the carboxy terminus of the protein, accumulated at least 10 times more immediate-early region 1 RNA than wild-type pSVCC2 did. In contrast, normal levels of delta S12-specific RNA were detected in cells cotransfected with wild-type pSVCC2. Therefore, the wild-type gene was capable of suppressing transcription of the mutant gene. Our results suggest that the wild-type major immediate-early protein of cytomegalovirus autoregulates transcription of immediate-early region 1 and that one of the regulatory domains is within the carboxy-terminal 145 amino acids of the viral protein.