O₂plays a pivotal role in aerobic metabolism and regulation of cell and tissue function. Local differences and fluctuations in tissue O₂levels are well documented; however, the physiological significance of O₂microgradients, particularly at the subcellular level, remains poorly understood. Using the cell-penetrating phosphorescent O₂probe Pt-Glc and confocal fluorescence microscopy, we visualized O₂distribution in individual giant (>100-μm) umbrella cells located superficially in the urinary bladder epithelium. We optimized conditions for in vivo phosphorescent staining of the inner surface of the mouse bladder and subsequent ex vivo analysis of excised live tissue. Imaging experiments revealed significant (≤85 μM) and heterogeneous deoxygenation within respiring umbrella cells, with radial O₂gradients of up to 40 μM across the cell, or ∼0.6 μM/μm. Deeply deoxygenated (5–15 μM O₂) regions were seen to correspond to the areas enriched with polarized mitochondria. Pharmacological activation of mitochondrial respiration decreased oxygenation and O₂gradients in umbrella cells, while inhibition with antimycin A dissipated the gradients and caused gradual reoxygenation of the tissue to ambient levels. Detailed three-dimensional maps of O₂distribution potentially can be used for the modeling of intracellular O₂-dependent enzymatic reactions and downstream processes, such as hypoxia-inducible factor signaling. Further ex vivo and in vivo studies on intracellular and tissue O₂gradients using confocal imaging can shed light on the molecular mechanisms regulating O₂-dependent (patho)physiological processes in the bladder and other tissues.