Innate defense against microbial infection requires the action of neutrophils, which have cytoplasmic granules replete with antibiotic proteins and peptides. Bactericidal/permeability-increasing protein (BPI) is found in the primary granules of adult neutrophils, has a high affinity for lipopolysaccharides (or "endotoxins"), and exerts selective cytotoxic, antiendotoxic, and opsonic activity against gram-negative bacteria. We have previously reported that neutrophils derived from newborn cord blood are deficient in BPI (O. Levy et al., Pediatrics 104:1327-1333, 1999). The relative deficiency in BPI of newborns raised the possibility that supplementing the levels of BPI in plasma might enhance newborn antibacterial defense. Here we determined the effects of addition of recombinant 21-kDa N-terminal BPI fragment (rBPI₂₁) on the growth and tumor necrosis factor (TNF)-inducing activity of representative gram-negative clinical isolates. Bacteria were tested in citrated newborn cord blood or adult peripheral blood. Bacterial viability was assessed by plating assay, and TNF- release was measured by enzyme-linked immunosorbent assay. Whereas adult blood limited the growth of all isolates except Klebsiella pneumoniae, cord blood also allowed logarithmic growth of Escherichia coli K1/r and Citrobacter koseri. Bacteria varied in their susceptibility to rBPI₂₁'s bactericidal action: E. coli K1/r was relatively susceptible (50% inhibitory concentration [IC₅₀], ~10 nM), C. koseri was intermediate (IC₅₀, ~1,000 nM), Klebsiella pneumoniae was resistant (IC₅₀, ~10,000 nM), and Enterobacter cloacae and Serratia marcescens were highly resistant (IC₅₀, >10,000 nM). All isolates were potent inducers of TNF- activity in both adult and newborn cord blood. In contrast to its variable antibacterial activity, rBPI₂₁ consistently inhibited the TNF-inducing activity of all strains tested (IC₅₀, 1 to 1,000 nM). The antibacterial effects of rBPI₂₁ were additive with those of a combination of conventional antibiotics typically used to treat bacteremic newborns (ampicillin and gentamicin). Whereas ampicillin and gentamicin demonstrated little inhibition of bacterially induced TNF release, addition of rBPI₂₁ either alone or together with ampicillin and gentamicin profoundly inhibited release of this cytokine. Thus, supplementing newborn cord blood with rBPI₂₁ potently inhibited the TNF-inducing activity of a variety of gram-negative bacterial clinical pathogens and, in some cases, enhanced bactericidal activity. These results suggest that administration of rBPI₂₁ may be of clinical benefit to neonates suffering from gram-negative bacterial infection and/or endotoxemia.