To report vitreous oxygen tension before, immediately after, and at longer times after vitrectomy. A prospective, interventional consecutive case series. Oxygen was measured using an optical oxygen sensor in patients undergoing vitrectomy. Intraoperatively, oxygen measurements were taken before and after vitrectomy in two intraocular locations: adjacent to the lens and in the mid-vitreous. Sixty-nine eyes underwent oxygen tension measurements at the time of vitrectomy. In baseline eyes, oxygen tension in the vitreous was low, measuring 8.7 +/- 0.6 mm Hg adjacent to the lens and 7.1 +/- 0.5 mm Hg in the mid-vitreous. The difference between the two locations was statistically significant (P < .003), indicating that vitreous gel maintains an intraocular oxygen gradient. Immediately after vitrectomy, oxygen tension in the fluid-filled eye was higher, measuring 69.6 +/-4.8 mm Hg adjacent to the lens and 75.6 +/- 4.1 mm Hg in the mid-vitreous. There was no statistically significant oxygen gradient between the two locations. The difference in oxygen tension pre- and postvitrectomy is highly statistically significant (P < .0001 lens, P < .0001 mid-vitreous). In eyes with a history of vitrectomy and previous removal of the vitreous gel, the intraocular oxygen tension was significantly higher than in eyes with a formed vitreous gel undergoing a first vitrectomy (P < .02 lens, P < .003 mid-vitreous). Vitrectomy surgery significantly increases intraocular oxygen tension during and for prolonged periods after surgery. This exposes the crystalline lens to abnormally high oxygen and may lead to nuclear cataract formation.