Newborns are at increased risk of overwhelming infection, yet the mechanisms underlying this susceptibility are incompletely defined. In this study we report a striking 1- to 3-log decrease in sensitivity of monocytes in human neonatal cord blood, compared with monocytes in adult peripheral blood, to the TNF-α-inducing effect of multiple TLR ligands, including bacterial lipopeptides (BLPs), LPS, and the imidazoquinoline compound, imiquimod. In marked contrast, TNF-α release in response to R-848, a TLR ligand that is a congener of imiquimod, was equivalent in newborn and adult blood. Differences in ligand-induced TNF-α release correlated with divergent ligand-induced changes in monocyte TNF-α mRNA levels. Newborn and adult monocytes did not differ in basal mRNA or protein expression of TLRs or mRNA expression of functionally related molecules. Newborn monocytes demonstrated diminished LPS-induced, but equivalent R-848-induced, phosphorylation of p38 mitogen-activated protein kinase and altered BLP- and LPS-induced acute modulation of cognate receptors, suggesting that the mechanism accounting for the observed differences may be localized proximal to ligand recognition by surface TLRs. Remarkably, newborn plasma conferred substantially reduced BLP-, LPS-, and imiquimod-induced TNF-α release on adult monocytes without any effect on R-848-induced TNF-α release, reflecting differences in a plasma factor(s) distinct from soluble CD14. Impaired response to multiple TLR ligands may significantly contribute to immature neonatal immunity. Conversely, relative preservation of responses to R-848 may present unique opportunities for augmenting innate and acquired immunity in the human newborn.