Background Despite major advances in the management of newborn infants, neonatal sepsis (NS) remain important causes of neonatal morbidity and mortality in the newborn, mainly among preterm and low birth weight infants. Objective: The aim of this study was to investigate the usefulness of neutrophil CD64 expression alone and together with other infection markers in NS. Methods: Peripheral blood samples were taken from 109 neonates, who were categorized into three groups: proven or clinical sepsis (n = 35); disease without infection (n = 42); and healthy controls (n = 32). Complete blood count with differential, interleukin-6 (IL-6), C-reactive protein (CRP), and cell surface expression of CD64 on neutrophils have been evaluated in a prospective manner as a diagnostic aid for NS. Results: Expression of CD64 was significantly enhanced in neonates with sepsis compared with newborns with disease without infection and healthy controls (P = 0.001 and P = 0.001, respectively). Cutoff values of IL-6, CRP, CD64(MFI), and CD64(i) were 24.9 pg/ml, 4.05 mg/l, 87.7, and 4.39, respectively. Sensitivity-negative predictive values of IL-6, CRP, and CD64(MFI)/CD64(i) were 80.0-90.6%, 80.0-88.8%, and 88.6-94.0%, respectively. Combining all three tests increased the sensitivity to 100%; however, specificity and positive predictive value decreased to 62.1 and 55.5%, respectively. Conclusions: CD64 might be used either alone or combined with IL-6 and CRP for early diagnosis of NS. The advantages of CD64 when compared with IL-6 and CRP are rapid quantitation, very small blood volume required, and easy handling. J. Clin. Lab. Anal. 24:363-370, 2010. (C) 2010 Wiley-Liss, Inc.