The expression of tumor necrosis factor alpha (TNF alpha) was examined in infants with leukomalacia by means of immunohistochemical methods with an antihuman TNF alpha monoclonal antibody. We studied 23 patients with neonatal leukomalacia, classified as having "focal," "widespread," or "diffuse" disease according to the distribution of the lesions, and 18 age-matched controls. TNF alpha immunoreactivity was positive in 19 of the 23 (83%) patients with leukomalacia, and in 7 of the 18 (39%) controls. TNF alpha was expressed mainly in glial cells in the deep white matter in both groups, and was most abundant around the necrotic foci in the focal group. TNF alpha immunoreactivity appeared earlier in patients with leukomalacia than in controls, being first detected at 25 and 29 weeks gestation, respectively. Immunofluorescence double-labeling revealed the TNF alpha -immunoreactive cells were Ricinus communis agglutinin-1 (RCA-1)-positive microglial cells. Thus, our study revealed increasing expression of TNF alpha in the normally developing brain during the late fetal period, and overproduction of TNF alpha by microglial cells associated with the pathogenesis of neonatal leukomalacia.