Matrix vesicles (MV) are microstructures localized to the extracellular matrix of developing hard tissues that induce mineral formation. MV proteins are not well characterized, and little is known of how they interact with the surrounding matrix. However, recent electron microscopic studies indicate that MV interact with matrix proteins in growth plate cartilage. In the studies now reported, procedures developed for dissecting various components from isolated MV led to the discovery that two major vesicle proteins (38 and 46 kDa) are readily released from MV by low ionic strength solutions. These low ionic strength-soluble proteins (LISSP) were shown to be major fragments of the link protein (LP) and hyaluronic acid-binding region (HABR) of matrix proteoglycans: they react immunologically with highly specific monoclonal antibodies to LP and HABR, and the NH2-terminal sequence of the 38-kDa LISSP is essentially identical to residues 40-78 of chicken cartilage LP and that the 46-kDa LISSP represents HABR. Release of both LISSP is enhanced by hyaluronidase treatment, indicating anchorage by a hyaluronate-mediated mechanism. Both LP and HABR are firmly attached to MV in either isotonic or hypertonic solutions. In contrast, our other studies show that dissociation of type II collagen from MV occurs only with hypertonic salts which do not release the LISSP. Thus, strong interactions occur under physiological conditions between MV and both the proteoglycans and collagens, but these take place by different mechanisms.