Background: Aging is associated with insulin resistance often attributable to obesity and inactivity. Recent evidence suggests that skeletal muscle insulin resistance in aging is associated with mitochondrial alterations. Whether this is true of the senescent myocardium is unknown. Methods: Twelve young (Y: 4years) and 12 old (O: 11 years) dogs, matched for body mass, were instrumented with LV pressure gauges, aortic and coronary sinus catheters, and flow probes on left circumflex artery. Prior to surgery, all dogs participated in a 6-week exercise program. Dogs underwent measurements of hemodynamics and plasma substrates before and during a two- hour hyperinsulinemic-euglycemic clamp to measure whole body and myocardial glucose and NEFA uptake. Following the protocol, myocardial and skeletal samples were obtained to measure components of the insulin-signaling cascade and mitochondrial structure. Results: There was no difference in plasma glucose (Y: 90±4; O: 87±4 mg/dl) but O had higher (p<0.02) non-esterified fatty acids (Y: 384±48; O: 952±97 µmol/L); and plasma insulin (Y: 39±11; O: 108±18 pmol/L). Old dogs had impaired total body glucose disposition (Y: 11.5±1; O: 8.0±0.5 mg/kg/min, p<0.05) and insulin stimulated myocardial glucose uptake (Y: 3.5±0.3; O: 1.8±0.3 mg/min/g, p<0.05). The impaired insulin action was associated with altered insulin signaling and GLUT-4 translocation. There were myocardial mitochondrial ultra-structural changes observed in association with decreased expression of UCP-3. Conclusion: Aging is associated with both whole body and myocardial insulin resistance, independent of obesity and inactivity, but involving altered mitochondrial structure and impaired cellular insulin action.