BACKGROUND:: Docosahexaenoic acid (DHA) and arachidonic acid (AA) are long-chain polyunsaturated fatty acids (LCPs) that play pivotal roles in growth and neurodevelopment. OBJECTIVE:: We aimed to quantify the synthesis of LCPs in preterm infants fed infant formula containing LCPs. DESIGN:: Twenty-two preterm infants were randomly assigned to either the no-LCP group (fed formula without LCPs; n = 11) or the LCP group (fed formula with LCPs; n = 11). Dietary LCPs had higher C content than did the endogenously synthesized LCPs, which were derived from linoleic and α-linolenic acids. The C content of major selected plasma fatty acids was measured by using gas chromatography-isotope ratio mass spectrometry at birth and at age 1, 3, and 7 mo. Absolute LCP synthesis and the percentage of LCP synthesis relative to dietary intake were calculated. RESULTS:: Percentage AA synthesis was 67.2 ± 7.8%, 35.9 ± 9.8%, and 29.0 ± 10.3%, and that of DHA was 41.7 ± 14.9%, 10.5 ± 8.1%, and 7.4 ± 6.2% at 1, 3, and 7 mo old, respectively. Absolute AA synthesis was 26.7 ± 4.2, 14.4 ± 3.9, and 11.6 ± 4.1 mg · kg · d and that of DHA was 12.6 ± 4.5, 3.2 ± 2.5, and 2.3 ± 1.9 mg · kg · d at 1, 3, and 7 mo old, respectively. AA and DHA synthesis decreased significantly (P < 0.01) with time, and AA synthesis was significantly (P < 0.01) greater than DHA synthesis. CONCLUSIONS:: By this novel approach, we measured endogenous LCP synthesis in infants receiving dietary LCPs over long periods. By age 7 mo, LCP synthesis was dramatically lower in preterm infants fed LCPs.