Two new neutral fac-[Re(CO)(3)(phen) L] compounds (1,2), with phen = 1,10-phenanthroline and L = O2C(CH2)(5)CH3 or O2C(CH2)(4)C CH, were synthetized in one-pot procedures from fac-[Re(CO)(3)(phen)Cl] and the corresponding carboxylic acids, and were fully characterized by IR and UV-Vis absorption spectroscopy, H-1-and C-13-NMR, mass spectrometry and X-ray crystallography. The compounds, which display orange luminescence, were used as probes for living cancer HeLa cell staining. Confocal microscopy revealed accumulation of both dyes in mitochondria. To investigate the mechanism of mitochondrial staining, a new non-emissive compound, fac-[Re(CO)(3)(phen)L], with L = O2C(CH2)(3)((C5H5)Fe(C5H4), i.e., containing a ferrocenyl moiety, was synthetized and characterized (3). 3 shows the same mitochondrial accumulation pattern as 1 and 2. Emission of 3 can only be possible when ferrocene-containing ligand dissociates from the metal center to produce a species containing the luminescent fac[Re(CO)(3)(phen)](+) core. The release of ligands from the Re center was verified in vitro through the conjugation with model proteins. These findings suggest that the mitochondria accumulation of compounds 1-3 is due to the formation of luminescent fac-[Re(CO)(3)(phen)](+) products, which react with cellular matrix molecules giving secondary products and are uptaken into the negatively charged mitochondrial membranes. Thus, reported compounds feature a rare dissociation-driven mechanism of action with great potential for biological applications.