The aim of this work is the characterization of interfaces in calcified tissues. In order to separate the non-collagenous bone proteins, according to their interaction with collagen or hydroxyapatite crystals, 10 sequential bone demineralizations using EDTA alone were carried out, followed by four sequential extractions using both EDTA and GuHCl. The extracts were characterized by SDS-PAGE, IR spectrum analysis, and kinetics of demineralization and proteins released. A great proportion of non-collagenous proteins are bound to the collagen matrix, many of which have a high affinity to it. This work demonstrates that collagen is not directly linked to the mineralized phase.