During infections, pathogens bind to toll-like receptor (TLR)4 and CD14 receptors and induce cytokine release, leading to inflammation. Here, we investigated TLR4 and CD14 expression on peripheral blood leukocytes (PBLs) and their roles in lipopolysaccharide (LPS)-induced cytokine and chemokine release. Full-term and preterm neonates and adults were studied. PBLs were pretreated with anti-TLR4- and anti-CD14-blocking antibodies and stimulated with LPS. Cytokine and chemokine levels were measured in supernatants. TLR4, CD14 expression, and LPS-induced CXCL8 release were higher in neonates, possibly contributing to aberrant inflammation. TLR4 blockade resulted in approximately 3-fold greater suppression of LPS-induced CXCL8 release in preterm neonates (38%) than in adults (14%). CD14 blockade (approximately 80%) in neonates induced approximately 3-fold greater inhibition of CXCL8 release, compared with anti-TLR4 (approximately 30%). Anti-TLR4 partly (50-60%) inhibited IL-10 and TNF-alpha, whereas anti-CD14 completely suppressed their release. Our findings reveal that neonates depend more on TLR4 for CXCL8 release. Furthermore, neonatal LPS-induced CXCL8 release, apart from TLR4/CD14-mediated signaling, is regulated by LPS interactions with other TLRs and/or immune receptors. IL-10 and TNF-alpha release depends on LPS binding not only to CD14/TLR4 but also to CD14 associated with another TLR. Our findings reveal the contribution of TLR4 and CD14 in neonatal cytokine and chemokine release and could aid in design of antagonists to prevent harmful inflammation.