Bradykinin (BK) or kallikreins activate B₂ receptors (R) which couple G_i and G_q proteins to release arachidonic acid (AA) and elevate [Ca²⁺]_i. Thrombin cleaves the protease-activated-receptor-1 (PAR1) that couples G_i, G_q and G_12/13 proteins. In CHO cells stably transfected with human B₂R, thrombin liberated little AA, but it significantly potentiated AA release by B₂R agonists. We explored mechanisms of cooperativity between constitutively expressed PAR1 and B₂R. We also examined human endothelial cells expressing both Rs constitutively. The PAR1 agonist hexapeptide (TRAP) was as effective as thrombin. Inhibitors of components of G_i, G_q and G_12/13 signaling pathways, and a PKC inhibitor, Go6976 blocked potentiation while phorbol, an activator, enhanced it. Several inhibitors, including a RhoA kinase inhibitor, a [Ca²⁺]_i antagonist, and an inositol-(1,3,4)-trisphosphate R antagonist, reduced mobilization of [Ca²⁺]_i by thrombin and blocked potentiation of AA release by B₂R agonists. Because either a non-selective inhibitor (isotetrandrine) of phospholipase A₂ (PLA₂) or a Ca²⁺-dependent PLA₂ inhibitor abolished potentiation of AA release by thrombin, while a Ca²⁺-independent PLA₂ inhibitor did not, we concluded that the mechanism involves Ca²⁺-dependent PLA₂ activation. Both thrombin and TRAP modified activation and phosphorylation of the B₂R induced by BK. In lower concentrations they enhanced it, while higher concentrations inhibited phosphorylation and diminished B2R activation. Protection of the N-terminal Ser¹-Phe² bond of TRAP by an aminopeptidase inhibitor made this peptide much more active than the unprotected agonist. Thus, PAR1 activation enhances AA release by B₂R agonists through signal transduction pathway.