High levels of CD40 ligand (CD40L) protein expression are induced on native T cells by increasing the intracellular Ca2+ concentration. In the present study we have shown that ionomycin induces CD40L gene transcription leading to mRNA accumulation which translates to high levels of protein expression. Conversely, agents which increase the intracellular levels of cyclic AMP (cAMP), such as prostaglandin E2 (PGE2) or dibutyryl cyclic AMP (dbcAMP), were unable to induce CD40L expression on T lymphocytes. Cell activation by phorbol 12-myristate 13-acetate (PMA) treatment had a slight effect on increasing CD40L mRNA and protein levels. However, PMA and dbcAMP synergized with ionomycin to significantly increase and to prolong the CD40L expression. Nuclear run-on assays revealed that PMA, but not dbcAMP, increased threefold the CD40L gene transcription rate induced by ionomycin. This effect was independent of de novo protein synthesis. In addition, at a posttranscriptional level, both reagents synergized with the Ca2+ ionophore to prolong the CD40L mRNA half-life by a mechanism which was also independent of de novo protein synthesis. Moreover, when transcription was blocked with actinomycin D, an increment of the CD40L transcript levels induced by PMA or dbcAMP on ionomycin-treated cells was observed in the presence of cycloheximide. This probably means that newly synthesized protein may contribute to the CD40L mRNA destabilization. In summary, these data show that PMA and dbcAMP synergized with ionomycin to increase the CD40L mRNA and protein levels. The up-regulatory effect of PMA was accomplished at a transcriptional and posttranscriptional level, whereas dbcAMP exerted its synergistic effect exclusively at a posttranscriptional level.