Apoptosis plays a key role in the development and maintenance of human tissues. This process has been studied traditionally in cells that are stained with exogenous fluorophores. These approaches affect cell viability, and thus are ill-suited for in vivo applications. We present an imaging approach that can identify apoptotic cells in living cell populations based on detection and quantification of distinct changes in the intensity and localization of cellular autofluorescence. Specifically, we acquire NAD(P)H, FAD, and redox ratio autofluorescence images of primary keratinocytes following 1, 9, 14, and 18 h of treatment with cisplatin, a known apoptosis-inducing chemotherapy agent. We find that intense autofluorescence combined with a low redox fluorescence ratio is progressively confined to a gradually smaller perinuclear cytoplasmic region with cisplatin treatment. Studies with exogenous nuclear fluorophores demonstrate that these autofluorescence changes occur at early stages of apoptosis. Additional costaining experiments suggest that this strongly autofluorescent, highly metabolically active perinuclear ring represents a subpopulation of mitochondria that are mobilized in response to the apoptotic stimulus and may provide the energy required to execute the final apoptotic steps. Thus, autofluorescence localization changes could serve as a sensitive, noninvasive indicator of early apoptosis in vivo.