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Firefly luciferase was shown to be completely folded and thus enzymatically active immediately upon release from the ribosome [Kolb et al. (1994) EMBO J. 13, 3631–3637]. However, no luciferase activity was observed while full‐length luciferase was attached to the ribosome as a peptidyl‐tRNA, probably because the C‐terminal portion of the enzyme is masked by the ribosome and/or ribosome‐associated proteins. Here we have demonstrated that the ribosome‐bound enzyme acquires the enzymatic activity when its C‐terminus is extended by at least 26 additional amino acid residues. The results demonstrate that the acquisition of the final native conformation by a nascent protein does not need the release of the protein from the ribosome.