Autocatalytic chromophore synthesis within the kindling fluorescent protein asFP595 is accompanied by a polypeptide chain break near the chromophore center. Tandem mass spectral analysis of the terminal proteolytic peptides adjacent to the asFP595 chain break allowed the elucidation of the exact structure of the asFP595 chromophore. The obtained results suggest that the mature asFP595 chromophore originates from a hydrolysis reaction of an intermediate acylimine-substituted structure.