IL-1 receptor antagonist (IL-1ra) is a cytokine that blocks the effects of IL-1 by binding to IL-1 receptors without inducing signal transduction. Amniotic fluid contains high concentrations of IL-1ra. The purpose of this study was 1) to analyze whether factors related to the mother or the fetus influence amniotic fluid IL-1ra concentration, and 2) to study whether the fetus is a source of IL-1ra. Two hundred two specimens of amniotic fluid, as well as 21 urine samples from newborn infants, were analyzed. Women carrying a female fetus had a higher concentration of amniotic fluid IL-1ra than those carrying a male fetus (female 136.4 ± 6.1 μg/L, n = 83; male 74.7 ± 3.7 μg/L, n = 119; p < 0.0001, unpaired two-sided t test). Length of gestation, presence or absence of labor signs, or elevated IL-1β in amniotic fluid did not affect the concentration of IL-1ra in amniotic fluid. Urine of infants taken during the first 48 h of life contained a high concentration of IL-1ra (91.1 ± 17.5 μg/L). The urinary IL-1ra concentration was higher in female newborns than in male newborns (females 124.0 ± 25.2 μg/L, n = 11; males 54.9 ± 19.1 μg/L, n = 10; p = 0.04). We conclude that 1) the concentration of IL-1ra in amniotic fluid and newborn urine is dependent on the gender of the fetus and of the newborn and 2) fetal urine is a major source of amniotic fluid IL-1ra. The higher IL-1ra in female-bearing gestations may contribute to the better resistance of female fetuses against preterm birth and perinatal infections.