Horse heart cytochrome c(cyt c) is a hemoprotein involved in two important cell events: the respiratory chain and the apoptosis. In the apoptosis process cyt c detaches from the inner mitochondrial membrane and attains the cytosol, but, because the protein is not fluorescent, it is difficult to follow its traffic inside the cells by fluorescence microscopy techniques. Therefore, the aim of the present work is to attach fluorescent 4-amino-1,8-naphthalimides (ANI) to the cyt c structure in a manner that preserves its native conformation. Coupling of the precursor 4-amino-1,8-naphthalic anhydride with cyt c was attempted by using three techniques: (i) incubation of the protein with the anhydride in molten imidazole (90ºC, during 5 min); (ii) sonication of the mixture in water; (iii) using a carbodiimide (EDAC) as coupling agent. According to spectrophotometric, chromatographic and mass spectrometric analysis, the use of molten imidazole seems to be the most efficient method to modify cyt c. ANI-modified cyt c did not exhibit significant structural alterations and potentially should maintain some biological properties. However, significant quenching of the attached imide fluorescence by the heme group was observed. The quenching intensity was attenuated by the association of the protein with membranes. These results point to the use of this modified fluorescent protein as a probe for membrane binding studies and perhaps for monitoring cell traffic.