This study shows that segmental expression of alkaline phosphatase (ALP) activity by the notochord of the Atlantic salmon (Salmo salar L.) coincides with the initial mineralization of the vertebral body (chordacentrum), and precedes ALP expression by presumed somite-derived cells external to the notochordal sheath. The early expression of ALP indicates that the notochord plays an instructive role in the segmental patterning of the vertebral column. The chordacentra form segmentally as mineralized rings within the notochordal sheath, and ALP activity spreads within the chordoblast layer from ventral to dorsal, displaying the same progression and spatial distribution as the mineralization process. No ALP activity was observed in sclerotomal mesenchyme surrounding the notochordal sheath during initial formation of the chordacentra. Our results support previous findings indicating that the chordoblasts initiate a segmental differentiation of the notochordal sheath into chordacentra and intervertebral regions.