Maturation of the pre-ribosomal RNA (pre-rRNA) in eukaryotes involves a series of processing steps that remove transcribed spacer sequences to produce mature ribosomes. In many organisms two processing sites are located in the 5'external transcribed spacer (5'ETS), along with the site that defines the 5'-end of the 18S rRNA. However, the pre-rRNA processing site near the start site of transcription, known as A', has long been believed to be absent in the single-celled yeast, Saccharomyces cerevisiae . Here we provide evidence that the A' pre-rRNA processing site is also present in the yeast 5'ETS, confirming conservation among single-celled and multicellular eukaryotes.