Purified bovine nasal cartilage proteoglycans (aggregate and subunit containing fractions) and to a lesser degree, chondroitin 4-sulfate of physiological size, retard seeded hydroxyapatite (HA) growth in vitro. The large hydrodynamic size and high charge density of these macromolecules are believed to be associated with the ability of proteoglycans to inhibit HA formation and growth. We now demonstrate the involvement of the negative charges of proteoglycans in this inhibition by comparing the inhibitory ability of chondroitin 4-sulfate and its desulfated analog, and by comparing the growth of HA seed crystals coated either with proteoglycan aggregates or chondroitin 4-sulfate to that of uncoated crystals. In the desulfation experiments, desulfated chondroitin sulfate was a less efficient HA growth inhibitor than untreated, undesulfated chondroitin sulfate of similar molecular size. Dextran sulfate showed higher inhibitory effectiveness than unchanged neutral dextran. Both experiments suggest that sulfate groups play an important role in the regulation of mineral deposition by proteoglycans. In the coating experiment, precoating of HA seed crystals with proteoglycan aggregates decreased the amount of HA precipitated as a function of time, suggesting proteoglycans may block the active nucleating sites on HA surface and slow down the growth process. Chondroitin 4-sulfate had a similar but weaker coating effect. Neutral dextran, having little affinity for HA, had no effect.