Slices of human annulus fibrosus were cultured under conditions that controlled their hydration and prevented loss of proteoglycans from the extracellular matrix. A quantitative analysis of proteoglycan synthesis was carried out. Both the absolute rate of synthesis and the topographical variation in chondrocyte activity changed with age; the most active cells in the adult were found in the mid-annulus region, whereas in the fetal disc the cells in the inner annulus were the most active. The conditions under which the tissue was stored, and changes in hydration during culture, had considerable effects on synthesis. Pathological discs had a wide range of biological activity that reflected the heterogeneous properties of these specimens. It is suggested that this culture method provides a means of investigating the way in which the synthesis of the macromolecular components of the intervertebral disc are coordinated and subsequently incorporated into the extracellular matrix.