Bioactive factors in human milk (HM) are crucial to the health of newborns, especially preterm infants. These compounds assist in reducing the oxidative stress that may occur as a result of combined exposure to supplemental oxygen and immature physiologic defenses. To identify the components in HM that contribute to its greater resistance to oxidative stress compared with infant formulae, enzymatic hydrolysates of HM were prepared, ultrafiltered, separated, and analyzed for antioxidant potential. The antioxidant activity [M Trolox equivalent (TE/g)] of nondigested milk, whole digested milk, and derived ultrafiltrates were 80.4 Ϯ 13.3, 159.0 Ϯ 5.6, and 127.4 Ϯ 3.1, respectively. An HPLC fraction denoted as fraction 23 (5274 Ϯ 630 M TE/g) was obtained and its constituents identified as tryptophan (Trp), peptides HNPI, and PLAPQA. Scavenging activity was not observed for PLAPQA, whereas moderate activity was associated with HNPI (144 Ϯ 10.7 M TE/g) and very high activity to Trp (7986 Ϯ 468 M TE/g). Trp addition to HM and two infant formulas significantly increased formulae antioxidant properties. Trp appeared to be a powerful free radical scavenger naturally present in HM. Its antioxidant effects and potential application in the diets of infants, particularly preterm, must be examined further. (Pediatr Res 66: 614-618, 2009)