Background: Unbound free fatty acids (FFAu) are the bioactive fraction of plasma free fatty acids (FFA). Most plasma FFA are bound to albumin. Only when FFA dissociate from albumin, do they become biologically active.Objective: To measure the first FFAu profiles in human infants and to measure these profiles before and during intravenous administration of the soybean lipid, intralipid (IL).Study design: The study population was 16 premature infants, from a parent study of 130 infants with birth weights 500–2000 g and gestational age 23–34 weeks. The infants chosen had plasma samples of ≥120 µL (volume needed for each FFAu profile measurement) in the first day of life. Infants received IL infusions starting in the second day of life at 1 g/kg/day, increasing by 1-g/kg/day daily up to 3 g/kg/day. FFAu profiles were determined during IL infusion when plasma was available. Profiles are the concentrations of the nine most abundant long-chain FFAu and were determined using novel fluorescent probes.Results: Before intralipid infusion unbound myristic acid was the dominant FFAu, as high as 78% of the total FFAu (sum of the 9 FFAu). In contrast, unbound linoleic acid was 0% in all infants. With increasing infusion of IL to 3 g/kg/day, unbound linoleic increased to 26% of the total FFAu, with unbound oleic, myristic, and linolenic acid the second, third and fourth most abundant. The average total FFAu concentration also increased from 4 nM before intralipid to 53 nM at 3 g/kg/day. During IL infusion the FFAu profiles approached the fatty acid composition of intralipid at 3 g/kg/day.Conclusions: This first study of FFAu profiles in neonates revealed that before IL infusion unbound linoleic acid was zero in all 16 infants and levels of myristic acid were exceptionally large, as much as 78% of the total FFAu profile. These results suggest important and previously unrecognized roles of lipid metabolism in early development. Zero unbound linoleic acid before IL infusion may help promote closure of the ductus arteriosus but after IL infusion, synthesis of arachidonic from linoleic acid may tend to promote patency. The high levels of unbound myristate may be needed for immediate neonatal energy needs.