OBJECTIVES: The 25-Item National Eye Institute Visual Function Questionnaire (VFQ-25) was designed to assess the influence of visual impairment on health-related quality of life (HRQL). The instrument has been developed and validated in samples of patients with various vision problems, but it has not previously been validated specifically for use in patients with diabetic retinopathy (DR). The purpose of this study was to assess the reliability and validity of the VFQ-25 in patients with DR. METHODS: Data were from a multicenter study on vision loss in patients with type 1 or type 2 diabetes and moderate to severe nonproliferative DR. Visual acuity was defined at each time point as each individual's best corrected vision as determined by the ETDRS visual acuity chart score. Patients completed the VFQ-25 and the SF-36. Psychometric analyses of baseline data examined internal consistency (Cronbach's alpha), criterion validity (Spearman correlations), and discriminant validity of the VFQ-25. RESULTS: The 684 enrolled patients were primarily Caucasian (77.8%) and male (63.3%), with a mean age of 59.3 years. Cronbach's alphas for VFQ-25 scales indicated good internal consistency, ranging from 0.72 to 0.93 for all subscales except two 2-item subscales, which had alphas of 0.68 (ocular pain) and 0.64 (social functioning). The VFQ-25 demonstrated convergent validity through statistically significant correlations with SF-36 subscales. For example, correlations of the VFQ-25 role difficulties subscale with the eight SF-36 subscales ranged from 0.25 to 0.35 (all p<0.0001). VFQ-25 subscales significant discriminated among groups of patients differing in ETDRS visual acuity scores (e.g., total VFQ-25 score = 79.1 for patients with ETDRS score of 48-78 letters; 83.0 for patients with 79-84 letters; 89.0 for patients with 85-100 letters). CONCLUSION: The VFQ-25 demonstrated adequate internal consistency reliability, criterion validity, and discriminant validity. Results support the use of this instrument among patients with diabetic retinopathy.