The rDNA cluster in the phytopathogenic fungus Ashbya gossypii consists of approximately 50 tandem repeat units of 8197 bp. Each unit carries a gene for the 35S pre-rRNA, processed into 18S, 5.8S and 25S rRNA, and a divergently transcribed gene for 5S rRNA. The well-characterized rDNA of the yeast Saccharomyces cerevisiae is the only other example of a completely sequenced rDNA unit (9137 bp) carrying both a 35S pre-rRNA and a 5S rRNA gene. The coding regions for the 5S, 5.8S, 18S and 25S rRNAs are 95-100% identical whereas transcribed and non-transcribed spacers show 43-66% sequence identity. Functionally characterized rDNA and rRNA elements of S. cerevisiae can be unambiguously recognized in the A. gossypii sequence, including the RNA polymerase-I transcription start site, two Reb1p enhancer binding sites and numerous recognition sequences for rRNA modification and processing. In addition to these functionally characterized sequences eight highly conserved elements from 10 to 71 bp were detected in the over 600-bp transcribed region upstream of the 18S rRNA gene which most likely play as yet uncharacterized functions at the DNA or RNA level. In addition to this work we started to identify A. gossypii homologs of S. cerevisiae nucleolar proteins involved in rDNA maturation.