Proteinase-activated receptor-1 (PAR(1)), a thrombin receptor and the prototype of a newly discovered G-protein-coupled receptor subfamily, plays an important role in tumor development and progression. In this study, we documented the expression of the thrombin receptors PAR(1), PAR(3), and PAR(4) in permanent hepatocellular carcinoma (HCC) cell lines and primary HCC cell cultures. Stimulation of HCC cells with thrombin and the PAR(1)-selective activating peptide, TFLLRN-NH(2), increased transmembrane migration across a collagen barrier. This effect was blocked by the PAR(1) antagonist SCH 79797, confirming that the PAR(1) thrombin receptor subtype is involved in regulating hepatoma cell migration. In addition, the PAR(4)-selective agonist, AYPGKF-NH(2), also stimulated HCC cell migration whilst the PAR(4) antagonist, trans-cinnamoyl-YPGKF-NH(2), attenuated the effect of thrombin on HCC cell migration. PAR(1)- and PAR(4)-triggered HCC cell migration was blocked by inhibiting a number of key mediators of signal transduction, including G proteins of the G(i)/G(o) family, matrix metalloproteinases, ERK/MAPKinase, cyclic AMP-dependent protein kinase, Src tyrosine kinase, and the EGF receptor kinase. Our data point to a cooperative PAR(1)/PAR(4) signaling network that contributes to thrombin-mediated tumor cell migration. We suggest that a combined inhibition of coagulation cascade serine proteinases, the two PARs and their complex signaling pathways may provide a new strategy for treating hepatocellular carcinoma.