The human rRNA promoter contains two distinct cis-control sequences, the core and upstream control element (UCE), that serve as the target for binding cellular trans-activating proteins involved in transcription initiation by RNA polymerase I. One of these factors, SL1, has been extensively purified and shown to be a species-specific factor required to reconstitute in vitro RNA synthesis. DNAase footprinting revealed that although SL1 alone does not bind specifically to rRNA promoter sequences, a second factor, UBF1, recruits SL1 to the template and directs binding to an extended region encompassing sequences in the UCE. Analysis of mutant and human-mouse hybrid promoters indicate that protein-DNA interactions at the UCE modulate the efficiency of rRNA synthesis. Transcription from the human rRNA promoter appears to require an unusual set of protein-DNA transactions in which recognition and binding to an upstream cis-control element is carried out by one factor (UBF1), whereas activation requires an additional factor, SL1, acting in conjunction with UBF1 to trigger transcription.