A yeast gene homologous to bacterial RNase III (RNT1) encodes a double-strand-specific endoribonuclease essential for ribosome synthesis. Two rRNA processing events are blocked in cells temperature sensitive for RNT1: cleavage at the snoRNA-dependent AO site in the 5 ETS and cleavage in the 3 ETS. Recombinant RNT1 protein accurately cleaves a synthetic 5 ETS RNA at AO site in vitro, in the absence of snoRNA or other factors. A synthetic 3 ETS substrate is specifically cleaved at a site 21 nt downstream of the 3 end 28S rRNA. These observations show that a protein endonuclease collaborates with snoRNAs in eukaryotic rRNA processing and exclude a catalytic role for snoRNAs at certain pre-rRNA cleavage.