The fatty acid composition of the membrane phospholipids phosphatidylcholine (PC) and phosphatidylethanolamine in insulin-sensitive Type I (soleus) and insulin-resistant Type II (EDL) muscle is not known. In the present studies, soleus and EDL muscles were removed from 250-300 g Sprague-Dawley rats, and the fatty acid composition of total and individual phospholipid (PL) species was quantitated. As expected, triglyceride content was increased twofold in soleus muscle. No quantitative differences in the individual PL species or cholesterol content were found between the two muscles. However, a striking difference in PL fatty acid composition was observed in the PC fraction. An increase in 16:0 with decreases in 18:0, 18:1, 22:5n-3, and 22:6n-3 (P < 0.001 for each) was observed in the PC fraction of EDL compared to that from soleus, consistent with reduced elongation of PC fatty acids. Inhibition of fatty acid oxidation with the carnitine palmitoyl transferase-1 inhibitor, etomoxir, did not alter the fatty acid pattern in either muscle. We conclude that an alteration in PL fatty acid composition consistent with reduced elongation of both saturated and unsaturated fatty acids is observed in Type II muscle. The restriction of these alterations to the PC fraction has important implications.