We hypothesized that improved glucose tolerance with rosiglitazone treatment would coincide with decreased levels of i.m. triacylglycerol (IMTG), diacylglycerol, and ceramide. Obese Zucker rats were randomly divided into two experimental groups: control (n = 9) and rosiglitazone (n = 9), with lean Zucker rats (n = 9) acting as a control group for obese controls. Rats received either vehicle or 3 mg/kg rosiglitazone for 6 wk. Glucose tolerance was impaired (P < 0.01) in obese compared with lean rats, but was normalized after rosiglitazone treatment. IMTG content was higher in obese compared with lean rats (70.5 +/- 5.1 vs. 27.5 +/- 2.0 micromol/g dry mass; P < 0.05) and increased an additional 30% (P < 0.05) with rosiglitazone treatment. Intramuscular fatty acid composition shifted toward a higher proportion of monounsaturates (P < 0.05) in obese rosiglitazone-treated rats due to an increase in palmitoleate (16:1; P < 0.05). Rosiglitazone treatment increased (P < 0.05) skeletal muscle diacylglycerol and ceramide levels by 65% and 100%, respectively, compared with obese rats, but elevated muscle diacylglycerol was not associated with changes in the total or membrane contents of the diacylglycerol-sensitive protein kinase C isoforms theta;, delta, alpha, and beta. In summary, we observed a disassociation among skeletal muscle IMTG, diacylglycerol and ceramide content, and glucose tolerance with rosiglitazone treatment in obese Zucker rats. Our data suggest, therefore, that rosiglitazone enhances glucose tolerance by mechanisms other than reduction of fatty acid accumulation within skeletal muscle.