Basal level transcriptional initiation of fission yeast ribosomal RNA genes is dependent on the core ribosomal RNA gene promoter and is stimulated by an upstream rDNA promoter element and by regulatory sequences located in its approximately 3.5 kb intergenic rDNA spacer. A Schizosaccharomyces pombe sequence-specific rDNA binding activity was characterized that interacted with the upstream rDNA promoter region and that associated with required RNA polymerase I transcription components in initial fractionation steps. The rDNA binding activity was further purified and found to specifically associate with a region of the rDNA promoter between -80 and -56. The promoter region required for stable binding correlates with that mediating activated levels of transcriptional initiation. This rDNA binding activity stimulates in vitro rRNA synthesis supported by templates bearing this upstream promoter domain but not by templates lacking it.