Prior studies suggest that the effect of nucleotide methylation can increase the stability of rRNA; however, the effect of such modifications on the structure or function of rRNA requires additional research. To further examine the effect of individual 2'-0-methylations during rRNA biogenesis, an expressed rRNA plasmid was used to analyze quantitatively the effects of base changes at three methylated sites in 25S rRNA of 'S. pombe'. Two of these sites, Uml942 and Am2307, are conserved among eukaryotes and one, Am2734, is only found in yeast cells. Complementary base pairs between the selected sites and their cognate snoRNAs were disrupted with site-specific mutations in the rRNA sequences to inhibit methylation in the target sites. The synthesis of two of the mutant rRNAs with changes at Uml942 and Am2734, respectively, was affected severely 'in vivo' as the RNAs appeared highly unstable and were degraded rapidly with little effect on cell growth. These results provide direct evidence to support the existing hypothesis that rRNA methylations can be critical sites for nuclease attack. To analyze these results genes encoding snoRNAs which methylate the three rRNA sites (Uml942, Am2307, and Am2734) also were isolated and cloned. Compensation mutations to complement the initial 25S rRNA changes were made in two of these snoRNAs, snR62 (for Uml942) and snR68 (for Am2734), as a preliminary step for future studies of complementation 'in vivo.'