N4-Acetylcytidine (ac4C) is a conserved RNA modification catalyzed in humans by the acetyltransferase NAT10. To profile RNA sites targeted by NAT10, Sas-Chen et al. developed ac4C-seq, which uses acid-catalyzed reactivity enhancement to reduce ac4C such that the resulting product can induce C-to-T misincorporation in reverse transcription and be read out in sequencing at single-nucleotide resolution. Using this method, the authors identified a common motif for NAT10 substrates and found that ac4C is distributed differently in eukaryotes and archaea. Ac4C is undetectable in eukaryotic mRNA but can be induced by overexpression of the NAT10 acetyltransferase complex. In hyperthermophilic archaea, ac4C exhibits pervasive distribution in all types of RNAs, and its abundance increases with temperature. Knocking out the NAT10 homolog in archaea induces decreased ac4C across all RNA types and reduced fitness at higher temperatures. Based on the structures of archaeal ribosomes, cytidine acetylation may stabilize the ribosome by replacing solvent molecules at higher temperatures. This study provides a useful tool for profiling cytidine acetylation and lays a foundation for further functional studies.