Tissue factor (TF) instigates the extrinsic pathway of blood coagulation. Plaque disruption and exposure of circulating factor VII/VIIa to subendothelial procoagulants such as TF leads to intravascular thrombosis, a frequent cause of acute atherosclerotic events. Although the expression of TF in the intima of human atherosclerotic lesions is well established, little is known about the mechanisms of TF regulation in vascular smooth muscle cells (SMC). We demonstrate here that TF colocalizes with the receptor CD40 on lesional SMC within atherosclerotic lesions in situ. In cultured vascular SMC, ligation of CD40 with native CD40 ligand (CD40L) derived from activated T lymphocytes or recombinant human CD40L (rCD40L) induced the transient expression of TF on the cell surface (as determined by FACS analysis) in a concentration- and time-dependent manner and enhanced total cell-associated TF (as determined by ELISA). CD40L-induced TF on vascular SMC is functional and activates coagulation. In accordance with the increased TF activity, stimulation of vascular SMC with rCD40L did not affect either protein expression or activity of tissue factor pathway inhibitors. In summary, these findings demonstrate the potential of the CD40/CD40L signaling pathway to augment the procoagulant activity in human vascular SMC. Because TF and CD40 colocalize on lesional SMC in human atheroma, CD40/CD40L signaling may contribute to the TF expression and hence to increased thrombogenicity of plaques during the inflammatory responses of atherogenesis and arterial injury.