A nonemissive cyclometalated iridium(III) solvent complex, without conjugation with a cell-penetrating molecular transporter, [Ir(ppy)(2)(DMSO)(2)](+)PF(6)(-) (LIrl), has been developed as a first reaction-based fluorescence-turn-on agent for the nuclei of living cells. LIrl can rapidly and selectively light-up the nuclei of living cells over fixed cells, giving rise to a significant luminescence enhancement (200-fold), and shows very low cytotoxicity at the imaging concentration (incubation time <10 min, LIrl concentration 10 mu M). More importantly, in contrast to the reported nuclear stains that are based on luminescence enhancement through interaction with nucleic acids, complex LIrl as a nuclear stain has a reaction-based mode of action, which relies on its rapid reaction with histidine/histidine-containing proteins. Cellular uptake of LIrl has been investigated in detail under different conditions, such as at various temperatures, with hypertonic treatment, and in the presence of metabolic and endocytic inhibitors. The results have indicated that LIrl permeates the outer and nuclear membranes of living cells through an energy-dependent entry pathway within a few minutes. As determined by an inductively coupled plasma atomic emission spectroscopy (ICP-AEC), LIrl is accumulated in the nuclei of living cells and converted into an intensely emissive adduct. Such novel reaction-based nuclear staining for visualizing exclusively the nuclei of living cells with a significant luminescence enhancement may extend the arsenal of currently available fluorescent stains for specific staining of cellular compartments.