The role of RNA methylation in the control of ribosome production in mammalian cells has been reinvestigated through a study of the effects in vivo of cycloleucine, a specific and reversible inhibitor of nucleic acid methylations. No close coupling is observed between methylation and transcription. During an extensive blocking of methylation, the synthesis of preribosomal 45-S RNA continues, although at a slightly reduced rate. Transcription and methylation can be temporarily uncoupled in vivo without impairing significantly the efficiency of the subsequent maturation of the transcript which takes place when the methylation is resumed. At the post-transcriptional level, two main observations are made. First, the cleavage pattern of ribosomal RNA is not qualitatively modified by the drug treatment. Preribosomal 45-S RNA which is synthesized during an extensive blocking of methylations (95%) is cleaved in vivo in a stepwise fashion into molecules resembling the species of the normal processing. Undermethylation does not result in the appearance of new size products and no extensive or rapid degredation of rRNA precursors can be detected. Second, the global efficiency of the process of ribosomal RNA maturation is severely affected, in quantitative terms; the undermethylation partially inhibits several stages in the maturation pathway and does not block selectively at a definite step of processing, as previously reported by others. Two main modifications are observed. (a) In the nucleus, the life times of the various undermethylated intermediates of ribosomal RNA maturation are markedly increased and a significant accumulation of these forms is detected by long-term labelling studies. (b) The rate of appearance of ribosomal 28-S RNA into the cytoplasm is severely affected (85-90% inhibition), in much higher proportions than the formation of its immediate precursor, nuclear 28-S RNA, thus suggesting a particular sensitivity to a lack of methylation of the last stage of preribosome maturation (possibly the release to cytoplasm). Finally, if a normal level of methylation does not appear to be stringently required for the completion of ribosomal RNA maturation, our results indicate that the degree of RNA methylation can modulate the general efficiency of the maturation processess.