Objective: To investigate vitreous levels of reactive oxygen species (ROS) in patients with proliferative diabetic retinopathy (PDR) and analyze ROS levels among different groups of PDR patients. Design: Retrospective case-control study. Participants: Thirty-nine eyes of 39 patients with PDR and 16 eyes of 16 non-PDR patients (control group) that underwent primary vitrectomy for complications of PDR and other conditions (control group), with a follow-up time 12 months. Methods: Proliferative diabetic retinopathy patients were classified into 3 groups according to the extent of fibrovascular proliferation: (1) no or focal adhesions at <= 3 sites (n = 17); (2) >= 1 broad adhesions or vitreous-retinal adhesions around disc, macula, and arcade (n = 12); and (3) vitreous-retinal attachment extending to the periphery or no posterior vitreous detachment with or without retinal detachment (RD) (n = 10). The control group (n = 16) contained non-PDR patients. Vitreous samples were obtained during measurement of vitrectomy and vitreous levels of ROS by luminol-enhanced chemiluminescence assay. Main Outcome Measures: Reactive oxygen species levels were recorded as mean (+/- standard deviation) chemiluminescence counts per 10 seconds. Correlations of vitreous levels of ROS among the 3 PDR groups and anatomic prognosis were evaluated. Multiple linear regression analysis of selective potential risk factors was performed to investigate the main determinants of ROS levels. Results: Vitreous ROS levels were significantly higher in patients with PDR (125.76 +/- 351.72 chemiluminescence counts per 10 seconds) than in control patients (0.37 +/- 0.72 chemiluminescence counts per 10 seconds; P < 0.0001). Reactive oxygen species levels were 1.86 +/- 1.63 (group 1), 24.47 +/- 22.68 (group 2), and 457.94 +/- 597.01 (group 3); the difference among groups was significant (P = 0.001). Regression analysis indicated that only patient grouping (according to the severity of fibrovascular proliferation) had a strong dependent association with ROS levels (P = 0.001). Final anatomic results revealed that recurrent untreatable RD occurred in 3 patients of group 3, who also had the highest ROS levels. Conclusions: Reactive oxygen species levels were significantly elevated in the vitreous fluid of PDR patients, and patients with a more advanced clinical PDR appearance had higher ROS levels. These findings suggest an association between ROS and the pathogenesis of PDR. Reactive oxygen species might be correlated with PDR severity.