The structure and extracellular assembly of the newly sythesized aggregating proteoglycans of the human intervertebral disc were examined using an explant culture system. The objective was to study the changes with aging, topography, and pathology, comparing newly synthesized with endogenous proteoglycans. No detailed studies of the biosynthesis of human disc proteglycans have been previously reported. A method of explant culture that minimizes swelling and matrix loss was used to maintain the tissue architecture. Slices of postmortem and pathologic disc tissues were incubated in medium containing polyethylene glycol at appropriate concentrations to balance the swelling pressure of the tissue. The disc slices were contained in small-pore dialysis tubing to prevent penetration of the polyethylene glycol into the tissue. The newly synthesized proteoglycans were radiolabled with [35S]-sulphate. Proteoglycans were then extracted from the tissue slices and characterized with gel chromatographic and electrophoretic techniques. It was found that a single, high molecular weight proteoglycan is the major 35S-labeled synthesis product of disc cells at all ages. However, biosynthetic changes do occur: the monomer made by fetal and newborn disc cells was larger than that of adults. Furthermore, adult disc cells made other minor large 35S-labeled products, the synthesis pattern of which varied between regions. These results provide the first evidence that biosynthetic changes contribute to the age-related increase in the heterogeneity of the human disc proteoglycan population.