Neonatal T cells, compared to adult memory/effector T cells, have a diminished capacity to generate most cytokines, with the exception of interleukin-2 (IL-2). Since most cytokine production is controlled at the level of transcription, we compared adult and neonatal T cells for their ability to transcribe two representative cytokines [IL-4 and CD40-ligand(CD40L)]. Cord blood (CB) and adult blood (AB) whole mononuclear cells were transiently transfected with reporter plasmids directed by the IL-2, IL-4, and CD40L proximal promoters, and then polyclonally activated in vitro. After correction for transfection efficiency, both cell types demonstrated similar levels of IL-2 promoter activity, whereas IL-4 and CD40L promoter activities in CB were approximately 50% as efficient as in AB. We have previously found that the nuclear factor of activated T cells-1 (NFAT1) transcription factor is limiting for IL-4 production by naïve AB CD4 T cells, and that functional NFAT binding sites are present in the CD40L and IL-4 promoters. Therefore, we analyzed CB T cells for NFAT expression based on DNA binding activity in gel shift assays and Western blotting. For gel shift studies, nuclear protein was isolated from CB or AB T cells after polyclonal activation, conditions which result in the cytoplasmic-to-nuclear translocation of NFAT proteins. We found a complex of nuclear protein from CB extracts, bound in vitro to an IL-4 promoter NFAT binding site, migrated with a faster mobility than the complex formed with AB T cell extracts. Moreover, this complex was not altered by antisera to NFAT1, while the majority of the complex from AB T cells was supershifted. Western blotting of total cellular protein revealed comparable levels of c-fos between AB and CB, but no detectable full length (115-130 kD) NFAT1 in the CB, as was present in the AB. This lack of NFAT1 expression correlated with reduced transcriptional activity mediated by a multimer of NFAT binding sites, when this construct was transfected into CB T cells and compared to AB T cells. These differences were specific in that the activities of AP-1 and NFκB multimer constructs in CB T cells were either similar to or greater than that in AB T cells. Increasing the intracellular levels of NFAT1, achieved by co-transfection of an NFAT1 cDNA expression plasmid, substantially increased IL-4 and CD40L expression in CB T cells. We conclude that NFAT1 is a likely candidate to limit the expression of CD40L and IL-4, and possibly other NFAT-dependent cytokines, by CB T cells.