The human cytomegalovirus UL34 gene represses expression of the US3 immune evasion gene and is essential for viral replication in cell culture. Two highly related proteins, an early and a late protein, are synthesized at different times during infection from the UL34 gene. The late protein differs from the early protein only by the omission of 21 amino terminal amino acids. Both UL34 proteins repress expression of the US3 promoter and bind specifically to a DNA element in the US3 gene. We have localized the DNA-binding domain of the UL34 open reading frame to amino acids 22 to 243. Surprisingly, this same region of the UL34 open reading frame was also sufficient for transcriptional repression of US3 expression. The UL34 gene is unusual in encoding proteins that have extensively overlapping DNA-binding and transcriptional regulatory domains.