The calcification of connective tissues, including cartilage, is under the control of many interacting systems. Proteoglycans are thought to retard the deposition of hydroxyapatite crystals, and modification of the proteoglycans presumably facilitates mineralization in those tissues that are actively calcifying. The mechanism underlying these regulations remains speculative. This study investigates this question by comparing the inhibitory effectiveness of several macromolecules at neutral pH and approximately physiological ionic strengths. Inhibitors tested include bovine nasal proteoglycan monomer A1D1D1 and aggregate-containing A1 fractions, glycosaminoglycan chains (chondroitin 4-sulfate), and neutral dextran (as an uncharged analog). Hydroxyapatite growth was assessed either by measuring the time-dependent decreases in solution calcium and phosphate concentrations, or by determining utilization of hydroxyl ion in a pH-Stat. All species studied inhibit hydroxyapatite growth, and the extent of inhibition for each class is concentration-dependent. The proteoglycan aggregate-containing A1 fraction is more effective than the proteoglycan monomer at the same concentration, and the proteoglycan monomer is more effective than chondroitin 4-sulfate. Neutral dextran inhibits hydroxyapatite growth less effectively than proteoglycans. These results suggest that inhibition of hydroxyapatite growth by proteoglycans critically depends on both status (aggregate, monomer, etc.) and hydrodynamic size of this macromolecule, supporting the hypothesis that modification of proteoglycans in vivo functions to modulate the effectiveness of proteoglycans as a hydroxyapatite growth inhibitor.