Despite the large differences in their length and nucleotide composition, comparative analyses of the internal transcribed spacer 1 (ITS1) of widely divergent eukaryotes have suggested a simple core structure consisting of a central extended hairpin and lesser hairpin structures at the maturing junctions [Lalev, A. I., and Nazar, R. N. (1998) J. Mol. Biol. 284, 1341-1351]. In this study, the ITS1 in the pre-rRNA transcripts of Schizosaccharomyces pombe cells was examined with respect to structural features that underlie rRNA maturation. When plasmid-associated rRNA genes were expressed in vivo, a deletion of any major hairpin structure significantly reduced or eliminated both small and large subunit RNAs. Only changes in the central extended hairpin or junction regions, however, entirely eliminated plasmid-derived RNAs or resulted in elevated precursor levels. Structure-disrupting base substitutions within the RAC protein complex binding site in the extended hairpin indicated that the secondary structure was critical for rRNA maturation; composition or other changes with respect to the binding site had only modest effects. A similar disruption at the junction with the 18S rRNA also had striking effects on rRNA maturation, including a highly elevated level of unprocessed precursor and a surprisingly critical effect on 5.8S rRNA production. As previously observed with the 3' external transcribed spacer, the results are consistent with a maturation mechanism in which an initial cleavage in the 5' junction region may be directed by the RAC protein complex. Although not critical to rRNA processing, analyses of termini based on S1 nuclease protection as well as cleavage studies, in vitro, with Pac1 ribonuclease raise the possibility that in eukaryotes, as previously observed in bacteria, the RNase III homologues normally initiate the separation of the subunit RNAs.