The green fluorescent protein (GFP) from the jellyfish Aequorea Victoria forms an intrinsic chromophore through cyclization and oxidation of an internal tripeptide motif [Prasher, D. C., et al. (1992) Gene 111, 229-233; Cody, C. E., et al. (1993) Biochemistry 32, 1212-1218]. We monitored the formation of the chromophore in vitro using the S65T-GFP chromophore mutant. S65T-GFP recovered from inclusion bodies in Escherichia coli lacks the mature chromophore, suggesting that protein destined for inclusion bodies aggregated prior to productive folding. This material was used to follow the steps leading to chromophore formation. The process of chromophore formation in S65T-GFP was determined to be an ordered reaction consisting of three distinct kinetic steps. Protein folding occurs fairly slowly (k(f) = 2.44 x 10(-3) s(-1)) and prior to any chromophore modification. Next, an intermediate step occurs that includes, but is not necessarily limited to, cyclization of the tripeptide chromophore motif (k(c) = 3.8 x 10(-3) s(-1)). The final and slow step (k(ox) = 1.51 x 10(-4) s(-1)) in chromophore formation involves oxidation of the cyclized chromophore. Since the chromophore forms de novo from purified denatured protein and is a first-order process, we conclude that GFP chromophore formation is an autocatalytic process.