The procoagulant thrombin promotes polymorphonuclear leukocytes (PMN) adhesion to endothelial cells by a mechanism involving expression of intercellular adhesion molecule-1 (ICAM-1) via an NF-B-dependent pathway. We now provide evidence that activation of c-Src is crucial in signaling thrombin-induced ICAM-1 expression via tyrosine phosphorylation of RelA/p65. Stimulation of human umbilical vein endothelial cells with thrombin resulted in a time-dependent activation of c-Src, with maximal activation occurring at 30 min after thrombin challenge. Inhibition of c-Src by pharmacological and genetic approaches impaired thrombin-induced NF-B-dependent reporter activity and ICAM-1 expression. Analysis of NF-B pathway revealed that the effect of c-Src inhibition occurred independently of IB degradation and NF-B DNA binding function and was not associated with exchange of NF-B dimers. Phosphorylation of RelA/p65 at serine 536, an event mediating the transcriptional activity of DNA-bound RelA/p65 was also insensitive to c-Src inhibition. Interestingly, thrombin induced association of c-Src with RelA/p65, and inhibition of c-Src prevented this response, indicating that this interaction is contingent upon activation of c-Src. We also observed that thrombin induced tyrosine phosphorylation of RelA/p65 and this phosphorylation was lost upon inhibition of c-Src, consistent with requirement of activated c-Src for interaction with RelA/p65. These data implicate an important role of c-Src in phoshorylating RelA/p65 to promote the transcriptional activity of NF-B and thereby ICAM-1 expression in endothelial cells.