CD154 (CD40 ligand (CD40L)) has been demonstrated to play an essential role in the development of humoral and cellular immunity through its interaction with CD40. While earlier studies have examined the regulation of CD154 expression by transcriptional and posttranslational pathways, scant data exist on its regulation at a posttranscriptional level. In this report we demonstrate that CD154 mRNA is rapidly turned over in primary culture of activated human T lymphocytes. Moreover, we demonstrate that CD154 mRNA is unstable, but can be stabilized by treatment with either phorbol esters or calcium ionophores. To address this lability of CD154 mRNA, we examined the ability of cytoplasmic proteins to bind to its 3' untranslated region (3'UTR). Two major proteins (p25 and p50) capable of binding the 3'UTR of CD154 were identified. The p25 binding activity was associated with polysomes and appeared to correlate with CD154 mRNA instability. Intriguingly, these proteins did not appear to bind to the AU-rich elements present in the 3'UTR of CD154. Rather, their binding was localized to unique sites between nt 471-811 of the 3'UTR, which lack any classical AU-rich elements. These data suggest that these proteins interact with distinct cis-acting elements that are important in the posttranscriptional regulation of CD154 expression. As such, identifying these proteins will help us understand the signals that are necessary for CD154 expression by activated T cells.