CX3CL1/fractalkine, a chemokine specific to monocytes and NK cells, is induced synergistically by TNF-alpha and IFN-gamma in vascular endothelial cells. However, the mechanism for this synergism remains unclear. This study explored the hypothesis that the CX3CL1 expression is regulated at a posttranscriptional level, which may responsible for the synergism between TNF-alpha and IFN-gamma. Brief exposure of HUVECs to TNF-alpha led to a robust increase in IFN-gamma-induced CX3CL1 production. We found that TNF-alpha stabilized CX3CL1 mRNA in HUVECs stimulated with IFN-gamma. Cloning of 3' untranslated region (UTR) of CX3CL1 mRNA revealed the presence of a single copy of nonametric AU-rich element in its 3'UTR, and a luciferase reporter assay showed that a single AU-rich element is a crucial cis-element in the posttranscriptional regulation of CX3CL1. TNF-alpha treatment resulted in the phosphorylation of p38 MAPK and its downstream target, MAPK-activated protein kinase-2, but IFN-gamma did not affect the levels of MAPK and MAPK-activated protein kinase-2 phosphorylation induced by TNF-alpha. Treatment of the cells with an inhibitor of p38 MAPK accelerated the decay of CX3CL1 mRNA induced by TNF-alpha or the combination of TNF-alpha and IFN-gamma. Immunoprecipitation assay revealed that mRNA stabilizer HuR directly binds to 3'UTR of CX3CL1 mRNA. CX3CL1 expression is under control of posttranscriptional regulation, which is involved in the synergistic induction of CX3CL1 in response to the combined stimulation with TNF-alpha and IFN-gamma.