Singlet oxygen, O(2)(a(1)Delta(g)), was produced upon pulsed-laser irradiation of an intracellular photosensitizer and detected by its 1275 nm O(2)(a(1)Delta(g)) -> O(2)(X(3)Sigma(-)(g)) phosphorescence in time-resolved experiments using (1) individual mammalian cells on the stage of a microscope and (2) suspensions of mammalian cells in a 1 cm cuvette. Data were recorded using hydrophilic and, independently, hydrophobic sensitizers. The microscope-based single cell results are consistent with a model in which the behavior of singlet oxygen reflects the environment in which it is produced;nevertheless, the data also indicate that a significant fraction of a given singlet oxygen population readily crosses barriers between phase-separated intracellular domains. The singlet oxygen phosphorescence signals reflect the effects of singlet-oxygen-mediated damage on cell components which, at the limit, mean that data were collected from dead cells and, in some cases, reflect contributions from both intracellular and extracellular populations of singlet oxygen. Despite the irradiation-induced changes in the environment to which singlet oxygen is exposed, the "inherent" intracellular lifetime of singlet oxygen does not appear to change appreciably as the cell progresses toward death. The results obtained from cell suspensions reflect key features that differentiate cell ensemble from single cell experiments (e.g., the ensemble experiment is more susceptible to the effects of sensitizer that has leaked out of the cell). Overall, the data clearly indicate that measuring the intracellular lifetime of singlet oxygen in a O(2)(a(1)Delta(g)) -> O(2)(X(3)Sigma(-)(g)) phosphorescence experiment is a challenging endeavor that involves working with a dynamic system that is perturbed during the measurement. The most important aspect of this study is that it establishes a useful framework through which future singlet oxygen data from cells can be interpreted.