Transcription from the human cytomegalovirus major immediate-early promoter is dependent on host-cell regulatory proteins. The interactions between cellular nuclear proteins and a unique sequence located from nucleotide position -660 to -540 was investigated. The unique region presents a defined target for multiple distinct DNA-binding proteins which appear, in part, to have overlapping binding sites. A minimum of five sequence-specific DNA-binding activities that interact with sequences between -632 and -602, -602 and -557, -602 and -590, -563 and -540, and -602 and -582 were detected. Evidence is presented to suggest that the -632 to -602 site, a previously characterized nuclear factor 1 binding site, does not bind NF1 but strongly interacts with a distinct cellular factor. The binding of cellular proteins to the unique sequence region was shown to be important in directing transcription from the major immediate-early promoter.