We have cloned zebrafish focal adhesion kinase (Fak) and analyzed its subcellular localization. Fak protein is localized at the cortex of notochord cells and at the notochord-somite boundary. During somitogenesis, Fak protein becomes concentrated at the basal region of epithelial cells at intersomitic boundaries. Phosphorylated Fak protein is seen at both the notochord-somite boundary and intersomitic boundaries, consistent with a role for Fak in boundary formation and maintenance. The localization of Fak protein to the basal region of epithelial cells in knypek;trilobite double mutant embryos shows that polarization of Fak distribution in the somite border cells is independent of internal mesenchymal cells. In addition, we show that neither Notch signaling through Suppressor of Hairless (SuH) nor deltaD is necessary for the wild-type segmental pattern of fak mRNA expression in the anterior paraxial mesoderm. However, nonsegmental expression of fak mRNA occurs with ectopic activation of Notch signaling through SuH and also in fused somite and beamter mutant embryos, indicating that there are multiple regulators of fak mRNA expression. Our results suggest that Fak plays a central role in notochord and somite morphogenesis.