A 5.45-kb fragment containing the 5' end of the ribosomal RNA transcriptional unit from the fission yeast Schizosaccharomyces pombe was cloned in the yeast-E. coli shuttle vector YEp13. The transcription start point was mapped by R looping and S1 nuclease protection. The sequence of the entire external transcribed spacer (ETS) and its flanking regions was determined. Comparison of the sequence around the transcription start point with those of four budding yeasts (Saccharomycetoideae) reveals a consensus sequence from position -9 to -4 from the start. This sequence is likely to be an important element of the promoter for yeast RNA polymerase I (Pol.I). Comparison of all known Pol.I promoter sequences reveals a strong bias for nucleotides (nt) at several positions between -16 and +10. These nt may have a critical role in the transcription initiation process. The S. pombe ETS, which comprises 1355 bp, is significantly longer than those of the budding yeasts and lacks any significant sequence homology with the Saccharomyces cerevisiae ETS. R-loop analysis reveals a putative processing site within the ETS of S. pombe.