OBJECTIVE: Endothelial dysfunction has been hypothesized as a possible pathogenic factor in the development of diabetic retinopathy (DR). We examined the relationship of DR to endothelium-dependent and endothelium-independent responses in skin microvascular flow. RESEARCH DESIGN AND METHODS: Participants consisted of 224 individuals with diabetes: 85 with type 1 diabetes and 139 with type 2 diabetes. Sodium nitroprusside (SNP) and acetylcholine (ACh) were delivered across the skin by iontophoresis. Laser Doppler flowmetry was used to assess the skin microcirculation response to SNP (endothelium-independent response) and ACh (endothelium-dependent response). The presence and severity of DR were graded from retinal photographs using a standard protocol. RESULTS: Of 224 participants, 64.3% had DR. After multivariable adjustment, participants with reduced responses to SNP or ACh were more likely to have DR, with an odds ratio (OR) of 2.33 (95% CI 1.09-5.01) for SNP and 2.20 (1.05-4.61) for ACh, comparing participants with responses below and above the median values. Participants with reduced responses (below the median) to both SNP and ACh were nearly four times more likely to have DR (OR 3.86 [1.45-10.3]) than those with SNP and ACh both above the median values. CONCLUSIONS: The presence of DR was associated with a reduction in skin microcirculation responses to iontophoresis of both SNP and ACh, suggesting that vascular processes associated with both endothelial dysfunction and endothelial function-independent mechanisms may be pathogenically related to DR.