N -acetylcytidine (ac C) is an ancient and highly conserved RNA modification that is present on tRNA and rRNA and has recently been investigated in eukaryotic mRNA . However, the distribution, dynamics and functions of cytidine acetylation have yet to be fully elucidated. Here we report ac C-seq, a chemical genomic method for the transcriptome-wide quantitative mapping of ac C at single-nucleotide resolution. In human and yeast mRNAs, ac C sites are not detected but can be induced-at a conserved sequence motif-via the ectopic overexpression of eukaryotic acetyltransferase complexes. By contrast, cross-evolutionary profiling revealed unprecedented levels of ac C across hundreds of residues in rRNA, tRNA, non-coding RNA and mRNA from hyperthermophilic archaea. Ac C is markedly induced in response to increases in temperature, and acetyltransferase-deficient archaeal strains exhibit temperature-dependent growth defects. Visualization of wild-type and acetyltransferase-deficient archaeal ribosomes by cryo-electron microscopy provided structural insights into the temperature-dependent distribution of ac C and its potential thermoadaptive role. Our studies quantitatively define the ac C landscape, providing a technical and conceptual foundation for elucidating the role of this modification in biology and disease .