The present study quantifies the relationships between diet fatty acid profile and fatty acid composition of rat skeletal muscle phospholipids. Young adult male Sprague–Dawley rats were fed, for 8 weeks, on one of twelve moderate-fat diets (25 % of total energy) differing only in fatty acid profile. SFA content ranged from 8–88 % of total fatty acids, MUFA 6–65 %, total PUFA 4–81 %, n-6 PUFA 3–70 % and n-3 PUFA 1–70 %. Diet PUFA included only essential fatty acids 18 : 2n-6 and 18 : 3n-3. The balance between n-3 and n-6 PUFA (PUFA balance) in the diet ranged from 1 : 99 to 86 : 14 % n-3 PUFA:n-6 PUFA. The slope of muscle phospholipid composition plotted against diet composition quantifies the response of muscle membrane composition to dietary fat (0, no response; 1, complete conformity with diet). The resulting slopes were 0·02 (SFA), 0·10 (PUFA), 0·11 (MUFA), 0·14 (n-3 PUFA) and 0·23 (n-6 PUFA). The response to PUFA balance was biphasic with a slope of 0·98 below 10 % diet PUFA balance and 0·16 above 10 %. Thus, low diet PUFA balance has greater influence on muscle composition than 18-carbon n-3 or n-6 PUFA individually. Equations provided may allow prediction of muscle composition for other diet studies. Diet PUFA balance dramatically affects muscle 20 : 4n-6 and 22 : 6n-3. This may have significant implications for some disease states in human subjects.