The fatty acid composition of phospholipids is an important determinant of membrane function. Although the mitochondria play a pivotal role in skeletal muscle function, the fatty acid composition of their individual phospholipids has not been examined. The purpose of this study was to determine the fatty acid profile of each phospholipid in rat skeletal muscle mitochondria and compare it with that of the whole muscle. Lipids were extracted from the gastrocnemius muscles of 10 Wistar rats, and phospholipids were separated by thin-layer chromatography. The fatty acid composition of each phospholipid was then determined by gas chromatography. The same procedure was applied to a mitochondrial preparation from these muscles. We found that the fatty acid composition of the individual mitochondrial phospholipids (phosphatidyl choline, phosphatidyl ethanolamine, cardiolipin, phosphatidyl inositol, phosphatidyl serine, sphingomyelin, and lysophosphatidyl choline) and of the total mitochondrial phospholipids differed markedly (P < 0.05) from the fatty acid composition of the corresponding whole muscle phospholipids. Notably, the mitochondrial phospholipids had higher percentages of MUFA [13.9 (2.1) vs. 10.3 (0.9)] and lower percentages of PUFA [34.8 (4.3) vs. 39.5 (5.2)] and n6 fatty acids [25.0 (2.5) vs. 27.6 (2.5)]. Overall, the mitochondrial phospholipids had a lower unsaturation index than whole muscle phospholipids [135 (20) vs. 161 (26)]. Because PUFA are susceptible to peroxidation, unlike saturated fatty acids and MUFA, we propose that the low polyunsaturation of mitochondrial phospholipids is the result of selective pressure toward membranes that are more resistant to oxidative damage by reactive oxygen species produced in their vicinity. The negative effect of the low polyunsaturation on membrane fluidity may be counterbalanced by the higher percentage of MUFA and the known low cholesterol content of mitochondrial membranes.