In one of the most extensive analyses to date we show that the balance of diet n−3 and n−6 polyunsaturated fatty acids (PUFA) is the most important determinant of membrane composition in the rat under ‘normal’ conditions. Young adult male Sprague–Dawley rats were fed one of twelve moderate-fat diets (25% of total energy) for 8weeks. Diets differed only in fatty acid (FA) profiles, with saturate (SFA) content ranging 8–88% of total FAs, monounsaturate (MUFA) 6–65%, total PUFA 4–81%, n−6 PUFA 3–70% and n−3 PUFA 1–70%. Diet PUFA included only essential FAs 18:2n−6 and 18:3n−3. Balance between n−3 and n−6 PUFA is defined as the PUFA balance (n−3 PUFA as % of total PUFA) and ranged 1–86% in the diets. FA composition was measured for brain, heart, liver, skeletal muscle, erythrocytes and plasma phospholipids, as well as adipose tissue and plasma triglycerides. The conformer–regulator model was used (slope=1 indicates membrane composition completely conforming to diet). Extensive changes in diet SFA, MUFA and PUFA had minimal effect on membranes (average slopes 0.01, 0.07, 0.07 respectively), but considerable influence on adipose tissue and plasma triglycerides (average slopes 0.27, 0.53, 0.47 respectively). Diet balance between n−3 and n−6 PUFA had a biphasic influence on membrane composition. When n−3 PUFA<10% of total PUFA, membrane composition completely conformed to diet (average slope 0.95), while diet PUFA balance>10% had little influence (average slope 0.19). The modern human diet has an average PUFA balance ~10% and this will likely have significant health implications. ► An extensive study of influence of diet fat on membrane composition in rat tissues. ► Membrane SFA, MUFA and PUFA homeostatically regulated irrespective of diet variation. ► Adipose tissue and plasma triglyceride composition are highly responsive to diet. ► Membranes respond biphasically to balance between diet n−3 and n−6 PUFA. ► Important health implications for diets with n−3 PUFA<10% total PUFA.