Transition metal complexes are versatile probes for cellular imaging and sensing. While the interactions of Ru(II) polypyridyl complexes with macromolecules such as DNA have long been studied, their application in cells or tissues has been limited by poor membrane permeability. Here, we report Ru(II) probes coupled to a membrane-permeable triphenylphosphonium (TPP) vector. The biocompatible conjugation was achieved through click chemistry of an azide-functionalized polypyridyl ligand with an alkyne-modified TPP moiety. Evidence of cellular localization, predominantly in the mitochondria, was derived from live-cell confocal fluorescence microscopy. These studies demonstrate that TPP-modified Ru(II) conjugates intercalate DNA, and so can potentially function as nucleic acid probes. Spectroscopic solution studies and luminescence imaging revealed that Ru-complexes containing a TPP-1,10-phenanthroline (TPP-phen) ligand display enhanced environmental stability over those with a TPP-dipyridophenazine (TPP-dppz) ligand, thereby highlighting the importance of probe design in biological applications.