Cyclometalated iridium(III) complexes have received considerable attention and are important candidates for use as luminescent probes for cellular imaging because of their potential photophysical properties. We previously reported that fac-Ir(atpy)(3) 4 (atpy = 2-(5'-amino-4'-tolyl)pyridine) containing three amino groups at the 5'-position of the atpy ligand shows a maximum red emission (at around 600 nm) under neutral and basic conditions and a green emission (at 531 nm) at acidic pH (pH 3-4). In this Article, we report on the design and synthesis of a new pH-sensitive cyclometalated Ir(III) complex containing a 2-(5'-N,N-diethylamino-4'-tolyl)pyridine (deatpy) ligand, fac-Ir(deatpy)(3) 5. The complex exhibits a considerable change in emission intensity between neutral and slightly acidic pH (pH 6.5-7.4). Luminescence microscopic studies using HeLa-S3 cells indicate that 5 can be used to selectively stain lysosome, an acidic organelle in cells. Moreover, complex 5 is capable of generating singlet oxygen in a pH-dependent manner and inducing the death of HeLa-S3 cells upon photoirradiation at 377 or 470 nm.