Phosphorescent heavy-metal complexes exhibit relatively long lifetimes and significant Stokes shifts for easy separation of excitation and emission and could be used as promising candidates for fluorescence probes. To date, however, very limited examples of the use of phosphorescent heavy-metal complexes in bioimaging have been reported. Herein, a series of cationic iridium(III) complexes, [Ir(dfpy)<inf>2</inf>L]<sup>+</sup>PF<inf>6</inf><sup>-</sup> (dfpy = 2-(2,4-difluorophenyl)pyridine;L denotes a series of N∧N ligands with different conjugated lengths), have been synthesized. By variation of the N∧N ligands, the emission colors of these complexes can be tuned from blue to red, while high quantum efficiencies are retained. Further investigation reveals that these complexes exhibit different excited-state properties, which are responsible for the significant emission color tuning. Moreover, the exclusive staining of cytoplasm and low cytotoxicity were observed for these cationic iridium(III) complexes, as well as good cell membrane permeability, which makes them promising candidates as fluorescent probes for living cell imaging. Furthermore, in order to tune the excitation wavelength to the longer region, another cationic iridium(III) complex with lower singlet and triplet energy levels was also investigated to be used as living cell imaging material, achieving a longer excitation wavelength of 488 nm. ©2010 American Chemical Society.