Opacification of the lens nucleus is a major cause of blindness and is thought to result from oxidation of key cellular components. Thus, long term preservation of lens clarity may depend on the maintenance of hypoxia in the lens nucleus. We mapped the distribution of dissolved oxygen within isolated bovine lenses and also measured the rate of oxygen consumption (QO2) by lenses, or parts thereof. To assess the contribution of mitochondrial metabolism to the lens oxygen budget, we tested the effect of mitochondrial inhibitors on QO2 and partial pressure of oxygen (PO2). The distribution of mitochondria was mapped in living lenses by 2-photon microscopy. We found that a steep gradient of PO2 was maintained within the tissue, leading to PO2 < 2 mmHg in the core. Mitochondrial respiration accounted for approximately 90% of the oxygen consumed by the lens, however, PO2 gradients extended beyond the boundaries of the mitochondria-containing cell layer, indicating the presence of non-mitochondrial oxygen consumers. Time constants for oxygen consumption in various regions of the lens and an effective oxygen diffusion coefficient were calculated from a diffusion/consumption model. Typical values were 3x10-5 cm2/s for the effective diffusion coefficient and a 5 minute time constant for oxygen consumption. Surprisingly, the calculated time constants did not differ between differentiating fibers (DF) that contained mitochondria and mature fibers (MF) that did not. Based on these parameters, DF cells were responsible for approximately 88% of lens oxygen consumption. A modest reduction in tissue temperature resulted in a marked decrease in QO2 and the subsequent flooding of the lens core with oxygen. This phenomenon may be of clinical relevance because cold, oxygen-rich solutions are often infused into the eye during intraocular surgery. Such procedures are associated with a strikingly high incidence of post-surgical nuclear cataract.