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Abstract: To elucidate the inhibition mechanisms of hydroxyapatite (HA), a biological model mimicking the mineralization process was developed. The addition of 4% (v/v) dimethyl sulfoxide (DMSO) in synthetic cartilage lymph (SCL) medium containing 2mM calcium and 3.42mM inorganic phosphate (Pi) at pH 7.6 and 37°C produced HA as matrix vesicles (MVs) under physiological conditions. Such a model has the advantage of monitoring the HA nucleation process without interfering with other processes at the cellular or enzymatic level. Turbidity measurements allowed us to follow the process of nucleation, whereas infrared spectra and X-ray diffraction permitted us to identify HA. Mineral formation induced by DMSO and by MVs in the SCL medium produced crystalline HA in a similar manner. The nucleation model served to evaluate the inhibition effects of ATP, GTP, UTP, ADP, ADP–ribose, AMP, and pyrophosphate (PPi). Here 10μM PPi, 100μM nucleotide triphosphates (ATP, GTP, UTP), and 1mM ADP inhibited HA formation directly, whereas 1mM ADP–ribose and 1mM AMP did not. This confirmed that the PPi group is a potent inhibitor of HA formation. Increasing the PPi concentration from 100μM to 1mM induced calcium pyrophosphate dihydrate. We propose that DMSO-induced HA formation could serve to screen putative inhibitors of mineral formation. [Copyright &y& Elsevier]