A cell-free translation system producing mature green fluorescent protein (GFP) can be a useful tool for studying the mechanism and kinetics of GFP chromophore formation, as well as for fast protein engineering. We report here that the mature GFP can be formed in the cell-free translation system from E. coli. The synthesis of GFP in the cell-free system reaches a plateau in 30 to 40 min whereas its maturation is completed in 4 h from the beginning of translation. The delay between the GFP synthesis and the chromophore formation in the cell-free system provides the possibility to isolate and to analyse maturation intermediates for elucidation of the modification pathway.