Abstract: Processing of the yeast pre-rRNA at site A(2) internal transcribed spacer 1(ITS1) has been shown to require several small nucleolar ribonucleoprotein particles (snoRNPs) as trans-acting factors. Here we report a detailed mutational analysis of the cid-acting signals required to specify the site of A(2) lie in the 3'-flanking sequence; deletion or substitution of nucleotides in this region strongly inhibits processing, and residual cleavage is inaccurate at the nucleotide level. In contrast, the deletion of the 5'- flanking nucleotides has no detectable effect on processing. An evolutionarily conserved sequence, ACAC, is located at the site of cleavage. Substitution of the 3' AC leads to heterogeneous cleavage, with activation of cleavage at an upstream ACAC sequence, In all mutants that retain an ACAC element, a site of cleavage is detected immediately 5' to this sequence, showing that this element is recognized. An ACAC sequence is, however, not essential for accurate cleavage of site A(2). An additional signal is also present 3' to A(2), in a region that has the potential to form a stem-loop structure that is evolutionarily conserved, but of low stability. As has been found for site A(1) (the 5' end of the yeast 18S rRNA), the identification of the site of processing at A(2) relies on multiple recognition elements.