The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a unique protein that emits green light due to a chromophore formed by oxidation of a specific amino acid sequence. This posttranslational modification occurs spontaneously or via ubiquitous enzymes. GFP was cloned and expressed in various organisms, showing that it becomes fluorescent without requiring specific enzymes. Mutagenesis revealed variants with altered fluorescence, including a blue-fluorescent mutant with histidine replacing tyrosine at position 66. These variants offer new possibilities for visualizing gene expression and protein localization in living cells. GFP's fluorescence is due to an internal chromophore, not a cofactor, and its properties can be modified through mutations. The study shows that GFP's fluorescence is formed through autoxidation, not requiring enzymes, and that its chromophore formation is a slow process with a time constant of about 4 hours. The ability to generate fluorescence in situ allows for continuous monitoring of gene expression and protein trafficking in living cells. The study also highlights the potential of GFP variants for two-color imaging and fluorescence resonance energy transfer. The findings suggest that GFP can be used as a reporter protein for monitoring promoter activity, although its slow fluorescence formation may limit its use for rapid changes. The chromophore of GFP is formed through oxidation of a specific sequence, and mutations can alter its excitation and emission properties, making it useful for various applications in molecular and cell biology.The green fluorescent protein (GFP) from the jellyfish Aequorea victoria is a unique protein that emits green light due to a chromophore formed by oxidation of a specific amino acid sequence. This posttranslational modification occurs spontaneously or via ubiquitous enzymes. GFP was cloned and expressed in various organisms, showing that it becomes fluorescent without requiring specific enzymes. Mutagenesis revealed variants with altered fluorescence, including a blue-fluorescent mutant with histidine replacing tyrosine at position 66. These variants offer new possibilities for visualizing gene expression and protein localization in living cells. GFP's fluorescence is due to an internal chromophore, not a cofactor, and its properties can be modified through mutations. The study shows that GFP's fluorescence is formed through autoxidation, not requiring enzymes, and that its chromophore formation is a slow process with a time constant of about 4 hours. The ability to generate fluorescence in situ allows for continuous monitoring of gene expression and protein trafficking in living cells. The study also highlights the potential of GFP variants for two-color imaging and fluorescence resonance energy transfer. The findings suggest that GFP can be used as a reporter protein for monitoring promoter activity, although its slow fluorescence formation may limit its use for rapid changes. The chromophore of GFP is formed through oxidation of a specific sequence, and mutations can alter its excitation and emission properties, making it useful for various applications in molecular and cell biology.