The focal adhesion kinase (FAK) and cell adhesion kinase beta (CAKbeta, PYK2, CADTK, RAFTK) are highly homologous FAK family members, yet clearly have unique roles in the cell. Comparative analyses of FAK and CAKbeta have revealed intriguing differences in their activities. These differences were investigated further through the characterization of a set of FAK/CAKbeta chimeric kinases. CAKbeta exhibited greater catalytic activity than FAK in vitro, providing a molecular basis for differential substrate phosphorylation by FAK and CAKbeta in vivo. Furthermore, the N terminus may regulate catalytic activity since chimeras containing the FAK N terminus and CAKbeta catalytic domain exhibited a striking high level of catalytic activity and substrate phosphorylation. Unexpectedly, a modulatory role for the N termini in subcellular localization was also revealed. Chimeras containing the FAK N terminus and CAKbeta C terminus localized to focal adhesions, whereas chimeras containing the N and C termini of CAKbeta did not. Finally, prominent changes in cell morphology were induced upon expression of chimeras containing the CAKbeta N terminus, which were not associated with apoptotic cell death, cell cycle progression delay, or changes in Rho activity. These results demonstrate novel regulatory roles for the N terminus of FAK family kinases.