Thiazolidinedione (TZD) insulin sensitizers are specific agonists of peroxisome proliferator activated receptor (PPAR)γ. However, their mechanism of action and the in vivo target tissue(s) that mediate insulin sensitization remain poorly defined. Although PPARγ messenger RNA expression has been reported in skeletal muscle, the expression of PPARγ within myocytes in intact muscle tissue has not been examined. An antipeptide PPARγ antibody was generated; immunohistochemistry was then used to demonstrate that PPARγ is present within nuclei of myocytes [in both skeletal (white and red fibers) and cardiac tissue (rodent and human)]. The effect of insulin sensitizer treatment on muscle insulin action was studied using ob/ob mice after 4 days dosing with a potent (6 nm PPARγ K_d) TZD (10 mg/kg·day). 2-deoxyglucose (2-DOG) uptake was then assessed in freshly isolated soleus muscles from lean vs. ob/ob vs. TZD-treated ob/ob mice. In lean mouse muscles, 2-DOG uptake was stimulated by 82%, 95%, 165% (with 25, 100, 2000 μU/ml insulin); muscles from ob/ob were severely insulin resistant (<80% stimulation with 2000 μU/ml insulin). Muscles from TZD-treated ob/ob displayed a normal insulin response with 100 (71%) or 2000 (158%) μU/ml insulin. Additional studies were performed using ZDF rats treated with/without TZD for 7 days. In vivo 2-DOG glucose uptake into soleus, gastrocnemius, and diaphragm muscles was measured during euglycemic-hyperinsulinemic clamp. Compared with lean rats, muscle 2-DOG uptake in ZDF was reduced by 52% (soleus) or 71% (diaphragm). Partial (40–60%) normalization of the reduced 2-DOG uptake was evident in TZD-treated ZDF rats. In contrast to the effect of in vivo treatment on muscle insulin action, preincubation of isolated soleus muscles from naive lean or ob/ob mice for 5 h with 100 nm TZD did not affect insulin-stimulated 2-DOG uptake. We conclude: 1) PPARγ is expressed in myocytes within skeletal and cardiac muscle. 2) In vivo activation of PPARγ by treatment of insulin-resistant mice/rats with a potent TZD corrects impaired muscle insulin action. 3) The lack of a direct effect on muscle after 5 h in vitro TZD incubation suggests that changes in insulin action may require a longer duration of PPARγ activation or that improved muscle insulin sensitivity may result from an indirect in vivo effect of PPARγ activation (e.g. changes in systemic lipid metabolism).