AbstractFatty acid composition of neutral and polar lipid fractions from rat hearts was determined in rats of different ages as their diet source changed. Piebald rats were weaned at 21 days and were fed standard lab chow. Lipids from rat hearts, mothers milk and lab chow were purified on a Sephadex G‐25 fine column and separated into neutral and polar lipid fractions by silicic acid column chromatography. These lipid fractions were then hydrolyzed and methylated with BF3 in methanol, prior to gas liquid chromatographic separation on a 1/8 in. × 10 ft aluminum column of 15% EGS on 80–100 mesh acid‐washed Chromosorb W. Three major fatty acids in the neutral lipid fraction comprised 72% of total neutral lipid fatty acids from young hearts. At sexual maturity (at least 74 days old) C18∶1 was the major fatty acid, followed by C16∶0 and C18∶0. The same three fatty acids comprised 83% of total polar lipid fatty acids, but C18∶0 was the major fatty acid, followed by C16∶0 and C18∶1. The fatty acid composition of dietary lipids influenced the total neutral lipid fatty acid composition of the rat heart, but had little influence on the fatty acid composition of the polar lipid fraction.