Typically, translation terminates when ribososmes encounter a stop codon. Stop codon readthrough results in carboxy-terminally extended nascent peptides, but its biological roles in eukaryotes have remained elusive. Here, Dunn et al. use a modified ribosome profiling assay to analyse genome-wide translation in Drosophila melanogaster. They confirm phylogenetically predicted readthroughs and identify ∼300 novel C-terminal extensions, suggesting that stop codon readthrough is prevalent. The authors found that readthrough is biologically regulated during development and that C-terminal extensions produce stable protein products. In addition, they show that their peptide sequences contain subcellular localization signals, which implies that readthrough can alter protein function. Importantly, readthrough also occurred in yeast and human foreskin fibroblasts, suggesting that it has a role in several organisms.