We recently showed that increased urinary excretion of the cross-linked, nonisomerized form of the C-telopeptide of collagen type I (alphaalphaCTX) could be a sensitive indicator of the presence of bone metastases in breast cancer patients. The present study was sought to investigate (a) the localization of alphaCTX epitopes in the proximity of a bone metastasis and (b) the relationship between number of metastases and the urinary excretion of alphaalphaCTX. Adjacent bone sections from breast cancer patients were stained for the presence of tumor cells (anti-cytokeratin antibody), osteoclasts (TRAcP activity), and alphaCTX (anti-alphaCTX antibody). The association between the extent of metastatic bone disease and urinary excretion of alphaalphaCTX measured with ELISA was assessed in 90 breast cancer patients (45 with bone metastasis and 45 without bone metastasis). Immunohistochemistry revealed accumulation of TRAcP-positive osteoclasts and intense staining for alphaCTX epitopes in the proximity of cytokeratin-positive bone metastasis. Areas of alphaCTX staining showed unstructured bone tissue under polarized light. In addition, there was a significant linear association between the number of bone metastases and the urinary levels of alphaalphaCTX in breast cancer patients with metastatic bone disease, independent of age and body mass index (r = 0.56, P