Objective: Leukocyte differentiation antigens are expressed on the cell membrane during activation. The purpose of this study was to evaluate leukocyte activation in premature neonates with sepsis. Paired blood samples from the same individual while sick and while convalescent were examined to quantify the expression of leukocyte antigens in these clinical states. Methods: Mononuclear blood cells from 21 premature infants (24 to 30 weeks’ gestation) were analyzed. The “sick” samples were drawn at the time of workup for sepsis; “convalescent” samples were drawn 20 days later. Samples were incubated with monoclonal antibodies to the lymphocyte antigens CD3, CD19, CD25, CD26, CD71, and CD69 and neutrophil antigens CD11b, CD11c, CD13, CD15, CD33, and CD66b. The cells were lysed, fixed, and analyzed by flow cytometry. Results: Twenty-one infants enrolled in the study had multiple sepsis evaluations and had more than one sample available for a paired observation. CD33, CD66b, and CD19 levels were significantly elevated in both the presumed sepsis and culture-proven sepsis groups when compared with the samples drawn from those same patients when healthy. Expression of CD33 and expression of CD66b were correlated, and in a multivariate analysis the elevation of antigen expression was predictive of sepsis. Conclusions: Leukocytes from preterm newborn infants respond to infection with an increased expression of CD19, CD33, and CD66b on their cell surfaces. (J Pediatr 2000;137:345-50)