CD40 ligand (CD40L) expression is now recognized to contribute in the pathogenesis of vascular disease. Because increased CD40L has been associated with myocardial infarction, effects of endothelial cells and cAMP with respect to CD40L regulation may be of clinical relevance. In the present study, endothelial cells are shown to markedly increase CD40L on naïve CD4⁺ T cells with a more modest effect on memory T cells. Furthermore, the addition of dibutyryl cyclic AMP (dbcAMP) synergistically increased naïve cell CD40L but inhibited memory cell CD40L. Although it has previously been recognized that human endothelial cells can increase T‐cell CD40L, this is the first description of the difference in responses of naïve and memory cells and the first demonstration of synergistic effects of endothelial cells and cAMP on CD40L regulation. Consistent with previous reports that CD40L regulation is distinctive, another marker of early activation (CD69) was not similarly regulated. The mechanisms of CD40L regulation were related to calcineurin and calcium/calmodulin dependent kinase IV (CaMKIV) signalling pathways. Endothelial cell costimulation of CD40L was found to be dependent upon calcineurin activity while cAMP actions to increase CD40L were dependent upon CaMKIV. Expression of a dominant negative CaMKIV construct further indicated an important role for CaMKIV in regulation of CD40L and cAMP responses. These data indicate that endothelial cell costimulation can interact with cAMP through calcium signalling pathways to synergistically enhance CD40L expression. Because increased CD40L is associated with atherosclerotic plaque and instability, results are relevant to the pathogenesis of atherosclerosis and myocardial infarction.