Background: Endothelial dysfunction precedes overt atherosclerosis and is present in patients with type 2 diabetes mellitus (T2DM). Myocardial perfusion imaging (MPI) is an effective method of detection of coronary artery disease (CAD); however, the relationship between endothelial function and MPI in asymptomatic patients with T2DM has not been examined. Methods and Results: This study used a subset of the population from the Detection of Ischemia in Asymptomatic Diabetics (DIAD) study. Endothelium-dependent vasodilation (EDV) and endothelium-independent vasodilation (EIV) were measured by use of brachial artery ultrasonography in 75 asymptomatic patients with T2DM (56 men; mean age, 58.6 ± 6.4 years; mean duration of diabetes, 8.4 ± 7.5 years) who underwent adenosine MPI. Of the patients, 15 (20%) had evidence of relative ischemia (MPI+) whereas 60 (80%) had a normal study (MPI-). Both EDV (3.5% ± 3.7% vs 4.5% ± 6.6%, P = not significant) and EIV (15.1% ± 7.5% vs 16.8% ± 8.4%, P = not significant) were similar in the 2 groups. On the basis of a receiver-operator analysis, an EDV response of 8% was selected as a cut point, with a negative predictive value of 93% (13/14 subjects with EDV ≥8% were MPI-). Conclusions: Endothelial function in asymptomatic patients with T2DM is not associated with the presence of relative myocardial ischemia by MPI; however, an EDV of 8% or greater has a high negative predictive value for the exclusion of CAD. © 2006 American Society of Nuclear Cardiology.