Like most eukaryotes, Saccharomyces cercvisiae cells contain a minor 5-8S(L) rRNA that, relative to the major 5.8Ss species, carries several extra nucleotides at the 5'-end. The two species are produced by alternative pathways that differ in the events removing the 3'-terminal region of Internal Transcribed Spacer 1 from the 27SA2 pre-rRNA. Whereas the pathway leading to 5.8S(S) rRNA is well established, that producing the 5'-end of 5.8S(L) (called B1(L)) is poorly understood. Northern analysis of two different mutants of S. cerevisiae that overproduce 5.8SL rRNA revealed the presence of a fragment corresponding to the 3'-terminal region of Internal Transcribed Spacer 1 (ITS1) directly upstream from site B1(L). Immunoprecipitation experiments showed this fragment to be associated with the trans-acting factor Rrp5p required for processing at the early sites A0-A3. Together these data clearly support that the 5'-end of 5.8SL rRNA is an endonucleolytic event. In vivo mutational analysis demonstrated the lack of any cis-acting sequence elements directing this cleavage within ITSI. (c) 2006 Elsevier Inc. All rights reserved.