Maturation of the large subunit rRNAs includes a series of cleavages that result in removal of the internal transcribed spacer (ITS2) that separates mature 5.8S and 25/28S rRNAs. Previous work demonstrated that formation of higher order secondary structure within the assembling pre-ribosomal particle is a prerequisite for accurate and efficient pre-rRNA processing. To date, it is not clear which specific sequences or secondary structures are required for processing. Two alternative secondary structure models exist for Saccharomyces cerevisiae ITS2. Chemical and enzymatic structure probing and phylogenetic comparisons resulted in one structure (Yeh & Lee, J Mol Biol, 1990, 211:699-712) referred to here as the "hairpin model." More recently, an alternate folded structure was proposed (Joseph et al., Nucleic Acids Res, 1999, 27:4533-4540), called here the "ring model." We have used a functional genetic assay to examine the potential significance of these predicted structures in processing. Our data indicate that elements of both structural models are important in efficient processing. Mutations that prevent formation of ring-specific structures completely blocked production of mature 25S rRNA, whereas those that primarily disrupt hairpin elements resulted in reduced levels of mature product. Based on these results, we propose a dynamic conformational model for the role of ITS2 in processing: Initial formation of the ring structure may be required for essential, early events in processing complex assembly and may be followed by an induced transition to the hairpin structure that facilitates subsequent processing events. In this model, yeast ITS2 elements may provide in cis certain of the functions proposed for vertebrate U8 snoRNA acting in trans.