Significance Translation termination is a crucial process during protein synthesis. Class I release factors (RFs) are in charge of recognizing stop codons and consequently hydrolyzing the peptidyl-tRNA at the ribosomal P site. High-resolution crystal- and cryo-EM structures of RFs bound to the ribosome revealed a network of potential interactions that is formed between the mRNA and RFs; however, it remained enigmatic which interactions are critical for accurate stop codon recognition and peptide release. By using chemically modified stop codon nucleotides, the importance and the contribution of single hydrogen bonds to stop codon recognition was investigated. This approach revealed a detailed picture of chemical groups defining a stop codon and contributing to the discrimination against sense codons during prokaryotic and eukaryotic translation termination.