Recent data indicate that peripheral insulin sensitivity may be influenced by dietary fat quality and skeletal muscle phospholipid fatty acid composition. During a health survey of 70-year-old men insulin sensitivity was measured by the euglycaemic hyperinsulinaemic clamp technique and the fatty acid composition of the serum cholesterol esters was determined (n = 215) by gas liquid chromatography. In a subsample the fatty acids of the skeletal muscle phospholipids and triglycerides were determined after fine needle biopsy from m. vastus lateralis (n = 39). The peripheral insulin sensitivity was significantly and negatively correlated to the proportion of palmitic (r = -0.31, p < 0.001), palmitoleic (r = -0.25, p < 0.001) and di-homo-gamma-linolenic (r = -0.33, p < 0.001) acids and positively to the content of linoleic (r = 0.28, p < 0.001) acid in the serum cholesterol esters. There was an even stronger negative relationship to the proportion of palmitic acid in the skeletal muscle phospholipds (r = -0.45, p < 0.004). The fatty acid composition was also significantly related to insulin sensitivity in a stepwise multiple regression analysis in the presence of other clinical variables, which were associated with insulin action in univariate analysis. Thus, more than 51% of the variation of the insulin sensitivity was explained by an equation containing body mass index, serum triglyceride concentration and the content of palmitic acid in the skeletal muscle phospholipids. It is concluded that the fatty acid composition in serum and of the phospholipids of skeletal muscle may influence insulin action in elderly men.