The focus of this study was to gain insight into the role(s) of osteonectin in the preferential metastasis of breast cancer cells to bone. Osteonectin was isolated from conditioned media of several cell lines including breast cancer (MDA-MB-435, MDA-MB-468), osteoblasts (hFOB1.19), non-neoplastic breast epithelial (hTERT-HME1), and vascular endothelial cells isolated from a bone biopsy (HBME-1). Chemical/physical properties of osteonectin from these five sources was analyzed to determine if unique configurations of osteonectin exist and therefore identify a chemotactic isoform. Osteonectin from all sources had a molecular weight of ∼46 kDa, N-linked glycosylation, and undetectable phosphorylated serines, sialic acids and O-linked oligosaccharides. The cDNA for osteonectin from the breast cancer, osteoblast, and breast epithelial cell lines was identical, while the vascular endothelial cell cDNA contained point mutations that resulted in eight amino acid substitutions. Bone-derived osteonectin was then analyzed to assess its influence on breast cancer cell motility and migration. Although osteonectin increased undirected MDA-MB-231 cell motility, it did not chemoattract the same breast cancer cell line. However, the breast cancer cells did migrate toward the known chemoattractant vitronectin and to bone extracts derived from wild-type and osteonectin-null mice. Migration to vitronectin was enhanced when osteonectin was also present. We concluded that osteonectin was not a chemotactic factor. However, through its anti-adhesive properties, osteonectin induced undirected breast cancer cell motility, and may have enhanced chemoattraction to vitronectin. J. Cell. Biochem. © 2005 Wiley-Liss, Inc.