The enzymology of RNase YI*, a recently discovered endoribonuclease from yeast, was studied and compared to other endonucleases for detection of single-strand regions and base pair mismatches in RNA. Its value for RNA structure analyses was assessed with Escherichia coli 5S rRNA as a model substrate. The generally accepted structure of the 5S rRNA is based on thermodynamic energy considerations as well as structures conserved in regions of the molecule during evolution. S1 and mung bean nucleases gave similar results with very marked preference only for the longest single-stranded region in the model. RNase YI* was much more discriminating for detecting unpaired nucleotides as well as short single-strand regions and predicted the generally accepted 5S rRNA structure. Preliminary experiments also indicated that RNase YI* was more sensitive than RNase I for detecting single or multiple base pair mismatches in an RNA-DNA hybrid.