Neonates are at increased risk of infections compared to adults. To dissect the mechanisms that contribute to neonatal immune deficiency, we compared MHC-II antigen processing and presentation by monocytes from umbilical cord blood and unrelated adult controls. Antigen-specific, co-stimulation-independent murine T hybridoma cells were used to detect peptide:HLA-DR complexes. Relative to adult monocytes, neonatal monocytes were significantly defective in processing and presentation of protein antigens and presentation of exogenous peptide. Defects in responses to protein antigens and exogenous peptide were of similar magnitude (56-81% decrease), indicating that the defect lies in antigen presentation as opposed to intracellular antigen processing. Average surface MHC-II levels on neonatal monocytes were 38% less than on adult monocytes. However, there was no correlation between decreased MHC-II expression on individual neonatal monocyte samples and reduced T cell responses. We demonstrate for the first time that neonatal monocytes are defective in MHC-II antigen presentation by a mechanism not correlated with decreased MHC-II expression.