In Trypanosoma brucei, alpha-amanitin-resistant transcription characteristic of RNA polymerase I is initiated at ribosomal RNA gene (RRNA), procyclin gene (GPEET or EP1), and variant surface glycoprotein gene expression site (VSG ES) promoters. The three promoter types do not share obvious sequence homologies, but contain a proximal domain I and a distal domain II within 80 bp upstream of the transcription initiation site. RRNA, GPEET and EP1, but not the VSG ES promoter, require additional upstream sequences for full activity. In the present study, we competed in-vitro transcription of circular template DNA with linear DNA fragments to identify promoter domains responsible for binding and sequestering essential trans-acting transcription factors. For the GPEET promoter, we found that domain III, located between positions -141 and -92, was most important for the DNA fragment to exert a transcription competition effect, whereas domain I, the only element absolutely required for transcription, was not. Moreover, insertions between promoter domains II and III reduced both transcription from the GPEET promoter and competition with the GPEET promoter fragment, suggesting that these two domains cooperate in the formation of a stable DNA-protein complex. Taken together, these results indicate a promoter structure very similar to that of the Saccharomyces cerevisiae RRNA promoter. In contrast, VSG ES promoter analysis showed that domains I and II are both necessary and sufficient to compete transcription. Despite this structural difference, our analysis provide evidence that GPEET and VSG ES promoters interact with a common factor that is also important for RRNA promoter transcription.