Tumor necrosis factor-alpha (TNF-alpha) is thought to be an important mediator in the pathogenesis of neonatal septicemia. To assess the role of TNF-alpha in neonatal sepsis, serum levels of TNF-alpha were measured in a group of neonates with septicemia and compared with the levels of gestational-postnatal, age-matched healthy controls. The relationships between severity of infection, the nature of causative microorganisms, and TNF-alpha levels were also investigated in this prospective study. A total of 49 infants (25 full-term, 24 preterm) with proven sepsis and 40 healthy infants (20 full-term, 20 preterm) were included. Serum TNF-alpha levels were measured using the TNF-alpha immunoradiometric assay. The median level of TNF-alpha was found to be significantly higher in infants suffering from sepsis (154 pg/mL) particularly in those with septic shock (242.5 pg/mL) as compared to healthy controls (61.5 pg/mL) (p < 0.001). No correlation was found between TNF-alpha and postnatal ages, gestational ages or birth weights of the infants. TNF-alpha levels were not different in surviving and terminal neonates. Although serum TNF-alpha levels were found to be slightly higher in gram-negative septicemia, the difference was not significant. These findings suggest that TNF-alpha plays an important role in the pathophysiology of neonatal septicemia, but its importance as a prognostic factor is not yet clear.