A variant of firefly luciferase in which the C-terminal end was extended with 44 amino acid residues served as a model protein in this study. After transcription and translation in vitro, the enzyme activity was measured when still attached to the ribosome and when released from the ribosome by incubation with RNase A or puromycin. It was found that the C-terminally extended luciferase already had activity when linked to the ribosome, but its activity was greatly increased when released from the ribosome. These results indicate that the luciferase is folded during synthesis on the ribosome; however, some conformational adjustments occur after its release from the ribosome which are required for the full expression of its enzymatic activity.