The expression of human cytomegalovirus (HCMV) genes during viral replication is precisely regulated, with the interactions of both transcriptional activators and repressors determining the level of gene expression. One gene of HCMV, the US3 gene, is transcriptionally repressed early in infection. Repression of US3 expression requires viral infection and protein synthesis and is mediated through a DNA sequence, the transcriptional repressive element. In this report, we identify the protein that represses US3 transcription as the product of the HCMV UL34 open reading frame. The protein encoded by UL34 (pUL34) binds to the US3 transcriptional repressive element in yeast and in vitro. pUL34 localizes to the nucleus and alone is sufficient for repression of US3 expression. The data presented here, along with earlier data (B. J. Biegalke, J. Virol. 72:5457-5463, 1998), suggests that pUL34 binding of the transcriptional repressive element prevents transcription initiation complex formation.