The green fluorescent protein (GFP) has emerged, in recent years, as a powerful reporter molecule for monitoring gene expression, protein localization and protein–protein interaction. Several mutant variants are now available differing in absorption, emission spectra and quantum yield. Here we present a detailed study of the fluorescence properties of the Phe-64→Leu, Ser-65→Thr mutant down to the single molecule level in order to assess its use in quantitative fluorescence microscopy and single-protein trafficking. This enhanced GFP (EGFP) is being used extensively as it offers higher-intensity emission after blue-light excitation with respect to wild-type GFP. By means of fluorescence spectroscopy we demonstrate the absence of the neutral form of the chromophore and the lack of photobleaching recovery after ultraviolet light irradiation. Furthermore, we show that the EGFP spectral properties from isolated to densely packed molecules are highly conserved. From these experiments EGFP emerges as an ideal molecule for quantitative studies of intra and intercellular tagged-protein dynamics and fluorescence-activated cell sorting, but not for monitoring single-protein trafficking over extended periods of time.