OBJECTIVE: To study insulin sensitivity and perfusion in skeletal muscle together with the beta-cell function in subjects with the m.3243A>G mutation in mitochondrial DNA, the most common cause of mitochondrial diabetes. RESEARCH DESIGN AND METHODS: We measured skeletal muscle glucose uptake and perfusion using positron emission tomography and 2-[18F]fluoro-2-deoxyglucose and [15O]H2O during euglycemic hyperinsulinemia in 15 patients with m.3243A>G. These patients included five subjects with no diabetes as defined by the oral glucose tolerance test (OGTT) (group 1), three with GHb <6.1% and newly found diabetes by OGTT (group 2), and seven with a previously diagnosed diabetes (group 3). Control subjects consisted of 13 healthy individuals who were similar to the carriers of m.3243A>G with respect to age and physical activity. Beta-cell function was assessed using the OGTT and subsequent mathematical modeling. RESULTS: Skeletal muscle glucose uptake was significantly lower in groups 1, 2, and 3 than in the control subjects. The glucose sensitivity of beta-cells in group 1 patients was similar to that of the control subjects, whereas in group 2 and 3 patients, the glucose sensitivity was significantly lower. The insulin secretion parameters correlated strongly with the proportion of m.3243A>G mutation in muscle. CONCLUSIONS: Our findings show that subjects with m.3243A>G are insulin resistant in skeletal muscle even when beta-cell function is not markedly impaired or glucose control compromised. We suggest that both the skeletal muscle insulin sensitivity and the beta-cell function are affected before the onset of the mitochondrial diabetes caused by the m.3243A>G mutation.