Thrombin, tumor necrosis factor (TNF)- and lipopolysaccharide (LPS) have each been implicated in endothelial cell and vascular smooth muscle cell (VSMC) activation. We wished to test the hypothesis that these three agonists display mediator and/or cell type-specific properties. The addition of thrombin to human pulmonary artery endothelial cells resulted in an upregulation of platelet-derived growth factor (PDGF)-A, tissue factor (TF), intercellular adhesion molecule (ICAM)-1 and urokinase (u-PA), whereas TNF- and LPS failed to induce PDGF-A. These effects were mimicked by PAR-1 activation. In VSMC, thrombin induced expression of TF and PDGF-A, but failed to consistently induce ICAM-1 or u-PA expression. In contrast, TNF- and LPS increased expression of all 4 genes in this cell type. Inhibitor studies in endothelial cells demonstrated a critical role for PKC in mediating thrombin, TNF- and LPS induction of ICAM-1, TF and u-PA; and p38 MAPK in mediating thrombin, TNF- and LPS induction of TF. Taken together, these results suggest that inflammatory mediators engage distinct signaling pathways and expression profiles in endothelial cells and VSMC. The data support the notion that endothelial cell activation is not an all-or-none phenomenon, but rather is dependent on the nature of the extracellular mediator.