Neonatal CD4+ T cells less CD154 protein and mRNA than adult CD4+ T cells after activation by calcium ionophore and phorbol ester, but the mechanism for this reduced expression and its relevance to the primary immune response remain unclear. We compared expression of CD154 protein and mRNA and CD154 gene promoter activity by purified naive (CD45RAhighCD45ROlow) neonatal and adult CD4+ T cells after activation by calcium ionophore (ionomycin) and phorbol myristate acetate (PMA) treatment or by engagement of ap TCR-CD3 complex. Substantial and consistent reductions in expression by neonatal cells were found in all cases and were paralleled by decreased CD154-dependent activation of a B cell line. CD69 expression by neonatal CD4+ T cells after ap TCR-CD3 engagement was also reduced compared to adult cells, which suggested that limitations in activation-induced signaling by neonatal CD4+ T cells occurred at a point upstream of where the signaling pathways leading to CD154 and CD69 expression diverge. Decreased CD154 expression by neonatal cells after αβ TCR-CD3 engagement was paralleled by a lower free intracellular calcium concentration, a key event for CD154 gene transcription. Reduced CD154 promoter activity by neonatal cells persisted when proximal signaling events were bypassed using ionomycin and PMA, suggesting an additional and more distal mechanism for decreased transcription. In contrast, CD154 mRNA stability was similar in neonatal and adult cells after either ionomycin and PMA stimulation or engagement of the αβ TCR-CD3 complex. We conclude that decreased CD154 production by neonatal CD4+ T cells is due to limitations in both proximal and distal activation events, which together ultimately limit CD154 gene transcription.