Feeding various dietary lipids did not alter the mass of phospholipids in rat heart mitochondria, but the phospholipids' fatty acid compositions changed. Although the compositional changes in mitochondrial phosphatidylcholine and phosphatidylethanolamine were negligible, linoleic acid [cis 18:2(n-6)] of cardiolipin was replaced by other fatty acids from dietary lipids. An analysis of the molecular species showed an even greater extent of change than was observed by fatty acid composition analysis. The quantity of 18:2(n-6)/18:2(n-6) molecular species [18:2(n-6) in C-1 position/18:2(n-6) in C-2 position] in cardiolipin decreased when rats were fed lipids that were depleted of an essential fatty acid but not deficient in essential fatty aids (eicosatrienoic acid did not appear). The decrease in 18:2(n-6)/18:2(n-6) species in cardiolipin was most pronounced in rats fed sardine oil, in which the ratio of (n-3) to (n-6) polyunsaturated fatty acids was 0.2. The O2 consumption rate of rat heart mitochondria decreased as the quantity of 18:2 (n-6)/18:2(n-6) cardiolipin species decreased.