An unusual property of ribosomal RNA transcription is the species specificity of promoter recognition. Unexpectedly, the sequence-specific RNA pol I transcription factors hUBF and xUBF, isolated from human and Xenopus cells, respectively, recognize the same DNA sequence elements. Despite this similarity in DNA binding activity, neither factor will functionally substitute for the other in reconstituted transcription assays, suggesting that the specificity of protein-DNA interactions cannot account for the species-specific activation of transcription by hUBF and xUBF. Interestingly, we find that hUBF and xUBF form distinctly different complexes with human SL1 at both the human and Xenopus promoters. Together these results strongly implicate specific protein-protein interactions between transcription factors as an important determinant of promoter selectivity and species specificity.