Objectives: To study whether early-onset neonatal sepsis is associated with a prenatal immune response with elevated umbilical plasma levels of inflammatory mediators, and to study whether mediator levels may be helpful in identifying infected neonates. Setting: Nested case-control study. Methods: Cord blood was sampled from 7,073 consecutively delivered neonates. After review of the medical records, neonates suspected to suffer from infection were classified as infected (n = 52) or noninfected but sick controls (n = 33). We also included a group of healthy controls (n = 99). Umbilical plasma levels of tumour necrosis factor-α (TNFα), interleukin (IL)-1β, IL-6, IL-8, soluble TNF receptors (p55 and p75), IL-1 receptor antagonist (IL-1RA) and C-reactive protein were measured by immunoassays. Results: Infected neonates had higher levels of TNFα, IL-1β, IL-6, IL-8, p55, p75 and IL-1RA than healthy controls (all p < 0.01). Among preterm infants (GA <37 weeks), those with infection (n = 11) had higher levels of IL-1β, IL-6, IL-8, p55 and p75 than noninfected sick controls (n = 13) (all p < 0.05), but among term infants, the infected did not differ from the noninfected sick controls. Receiver operator characteristic plots showed that IL-1β, IL-6 and IL-8 identified preterm infected neonates accurately. Conclusions: Early-onset neonatal sepsis is associated with a prenatal immune response with increased TNFα, IL-1β, IL-6, IL-8, p55, p75 and IL-1RA levels in umbilical plasma. Among neonates who present symptoms suggestive of infection, cytokine levels may be helpful in identifying preterm, but not term infected individuals.