A reverse-transcriptase stop, consistent with 2΄-O-methylation, was observed at nucleotide S1316 when the artificial snoRNA containing the human U24 C΄/D΄ motif (hU24) was expressed, but not in the absence of a snoRNA (Figure 1E; methylation bands indicated by asterisk). A weaker stop was frequently observed at nucleotide S1315 one nucleotide above the target site, as we have observed previously (24). It is important to note that we also observed a weak stop one nucleotide above almost all natural methylation sites in the rRNA that we tested. We believe that this additional, weaker band, is due to additional stalling by reverse transcriptase and does not reflect additional methylation. 2΄-O-methylation at nucleotide S1316 was also seen when snoRNAs containing the C΄/D΄ motifs of snR13 and U24 were expressed. In contrast, the snoRNA containing the U18 C΄/D΄ motif resulted in methylation of nucleotides S1316 and S1315 while the snR48 C΄/D΄ directed modification at sites S1316 and S1314 (Figure 1E). Our data clearly show that the C΄/D΄ motifs of U18 and snR48 directed the methylation of two distinct nucleotides in the target region. Indeed, the methylation pattern seen for the U18 and snR48 C΄/D΄ motifs replicated the 2΄-O-methylation pattern attributed to the full-length snoRNAs (Figure 1C and D). Surprisingly, while multiple-site methylation was not seen with the box C΄/D΄ snoRNA motifs of snR13 and U24, both motifs directed modification of a nucleotide six nucleotides upstream of the D΄ box (Figure 1D). Furthermore, our data suggest that for both U24 and snR13, other elements in the snoRNA, in addition to the C΄/D΄ motif, or possibly an additional trans-acting factor, play a role in the 2΄-O-methylation of multiple sites in their target region.