This study investigated the subcellular compartmentalization of focal adhesion kinase (FAK) fragments and their regulation during apoptosis of human umbilical vein endothelial cells. A 50 kDa NH2-terminal FAK fragment and a 120 kDa FAB variant were constitutively expressed and specifically found in the nuclear fraction of cells, while a 55 kDa COOH-terminal FAK fragment was only in the cytosolic fraction, FAK cleavage fragments generated during apoptosis remained in the cytosol, while p120FAK and p50 NH2-terminal FAK remained in the nuclear compartment, The caspase inhibitor, ZVAD-fmk, prevented the apoptosis-induced proteolysis of p125 and p120FAK, generation of the 80 kDa cleavage product, and increased expression of p50N-FAK. Western blot with phospho-specific FAB: showed that nuclear p125(FAK) was phosphorylated at a significant level at Y861, while FAB phosphorylated at Y397 and Y407 was largely in the cytosol. These results indicate that FAK NH2- and COOH-terminal domain fragments are segregated between nuclear and cytosolic compartments in endothelial cells and suggest novel functions for the FAK NH2-terminal domain.