Clinical evidence has indicated that the neonatal cell-mediated immune response to primary infection is delayed when compared to that of adults with the same primary infection. The mechanisms regulating the development of antigen-specific T-cell immunity in neonates remain to be elucidated. We examined the primary immune response to the non-recall antigen, keyhole limpet haemocyanin (KLH) in adults and neonates in vitro. We report here that conventional bulk culture methods show reduced proliferative responses in neonates although statistical significance was not achieved. Using limiting dilution analysis, the frequencies of KLH-specific T lymphocytes were 10–100-fold lower in neonates when compared to adults. Interleukin-2 (IL-2) production was significantly lower in the supernatants of neonatal mononuclear cells (MNC) stimulated with KLH when compared to adults. Addition of exogenous IL-2 increased precursor frequencies twofold in both adult and newborn cultures. In contrast to the secreted IL-2 levels, IL-2 mRNA expression was higher in antigen-stimulated neonatal MNC preparations, even though proliferation was lower. These observations indicate differential in vitro responsiveness in neonates and adults to primary antigenic challenge. Since no IL-2 was detected in cell lysates, the presence of high levels of IL-2 mRNA and low IL-2 production suggests inability by neonatal MNC to translate IL-2. This deficiency in IL-2 production may explain the reduced precursor frequencies, suggesting failure to recruit T lymphocytes in order to expand the KLH-specific T-cell response. These observations are important for the understanding of the development of primary immune responses and immunological maturation in neonates.