Neonates respond suboptimally to many vaccines. The reasons for this defect are unclear, but suboptimal antigen presentation by dendritic cells has been suggested as one possibility. In this report we describe an in vitro system that allows the generation of large numbers of resting murine neonatal dendritic cells facilitating their study. Using this system, we show a clear reduction in the ability of neonatal dendritic cells to present soluble ovalbumin, while the capacity to present ovalbumin peptide is intact. This suggests a specific defect in cross-presentation of exogenous antigen via the major histocompatibility complex (MHC) class I pathway. Deficient cross-presentation may contribute to the suboptimal CD8 T-cell response to vaccines in neonates.