The fatty acid-conjugated linoleic acid (CLA) enhances glucose tolerance and insulin action on skeletal muscle glucose transport in rodent models of insulin resistance. However, no study has directly compared the metabolic effects of the two primary CLA isomers, cis-9,trans-11-CLA (c9,t11-CLA) and trans-10,cis-12-CLA (t10,c12-CLA). Therefore, we assessed the effects of a 50:50 mixture of these two CLA isomers (M-CLA) and of preparations enriched in either c9,t11-CLA (76% enriched) or t10,c12-CLA (90% enriched) on glucose tolerance and insulin-stimulated glucose transport in skeletal muscle of the insulin-resistant obese Zucker (fa/fa) rat. Animals were treated daily by gavage with either vehicle (corn oil), M-CLA, c9,t11-CLA, or t10,c12-CLA (all CLA treatments at 1.5 g total CLA/kg body wt) for 21 consecutive days. During an oral glucose tolerance test, glucose responses were reduced (P < 0.05) by 10 and 16%, respectively, in the M-CLA and t10,c12-CLA animals, respectively, whereas insulin responses were diminished by 21 and 19% in these same groups. There were no significant alterations in these responses in the c9,t11-CLA group. Insulin-mediated glucose transport activity was enhanced by M-CLA treatment in both type I soleus (32%) and type IIb epitrochlearis (58%) muscles and by 36 and 48%, respectively, with t10,c12-CLA. In the soleus, these increases were associated with decreases in protein carbonyls (index of oxidative stress, r = -0.616, P = 0.0038) and intramuscular triglycerides (r = -0.631, P = 0.0028). Treatment with c9,t11-CLA was without effect on these variables. These results suggest that the ability of CLA treatment to improve glucose tolerance and insulin-stimulated glucose transport activity in insulin-resistant skeletal muscle of the obese Zucker rat are associated with a reduction in oxidative stress and muscle lipid levels and can be specifically ascribed to the actions of the t10,c12 isomer. In the obese Zucker rat, the c9,t11 isomer of CLA is metabolically neutral.