Functional regulation by cofactors is fundamentally important for the highly ordered, consecutive activation of the coagulation cascade. The initiating protease of the coagulation system, factor VIIa (VIIa), retains zymogen-like features after proteolytic cleavage of the activating Arg(15)-Ile(16) peptide bond and requires the binding of the cofactor tissue factor (TF) to stabilize the protease domain in an active enzyme conformation. Structural comparison of TF-bound and free VIIa failed to provide a conclusive mechanism for this catalytic activation. This study provides novel insight into the cofactor-dependent regulation of VIIa by demonstrating that the side chain of Phe(225), an aromatic residue that is common to allosterically regulated serine proteases, is necessary for optimal TF-mediated activation of VIIa's catalytic function. However, mutation of Phe(225) did not abolish the cofactor-induced stabilization of the Ile(16)-Asp(194) salt bridge, previously considered the primary switch mechanism for activating VIIa. Moreover, mutation of other residue side chains in the VIIa protease domain resulted in a reduced level of or no stabilization of the amino-terminal insertion site upon TF binding, with little or no effect on the TF-mediated enhancement of catalysis. This study thus establishes a crucial role for the aromatic Phe(225) residue position in the allosteric network that transmits the activating switch from the cofactor interface to the catalytic cleft, providing insight into the highly specific conformational linkages that regulate serine protease function.