Human endothelial cells (ECs) constitutively express OX40L and co-stimulate memory CD4(+) T cell proliferation that is dependent upon OX40-OX40L interaction. In vivo, OX40 prolongs T cell survival; however, an unanswered question is whether it can also prolong synthesis of proliferation-sustaining cytokines such as IL-2. Here we show that EC co-stimulation results in the secretion of T cell IL-2, IL-3 and IFN-gamma and that in the absence of OX40 signals synthesis largely ceases by 12-18 h, but is prolonged up to 60 h in the presence of OX40 signaling. Blocking OX40-mediated cytokine expression at later times suppresses T cell proliferation and this can be overcome by addition of exogenous IL-2. We find that OX40 signaling has discrete effects on T cell activation as it does not affect expression of IL-10, CD25, CD69 or soluble IL-2R. Also, OX40 does not appear to alter IL-2 transcription, but rather acts to stabilize a subset of cytokine mRNAs, increasing their half-lives by 3-6-fold. We further show that OX40L induces activation of p38 mitogen-activated protein kinase (MAPK) and phosphotidyl-inositol-3-kinase (PI3K) in T cells, and using specific inhibitors, we find that increased mRNA half-life is dependent upon both these pathways but is independent of c-jun-N-terminal kinase (JNK). Thus, EC co-stimulation through OX40 leads to prolonged T cell cytokine synthesis and enhanced proliferation.