Mammary tumors in female dogs are usually malignant and tend to metastasize to distant organs, especially to regional lymph nodes and lungs. Radiography is the standard diagnostic method to detect pulmonary metastases in these animals. Magnesium (Mg). vascular endothelial growth factor (VEGF), and osteopontin (OPN) levels have been used in recent studies to make prognoses of human breast cancer. To the best of our knowledge, however, there are not many studies that have been performed on this subject, and there is no study on animals in which the three indicators are scrutinized together. The aim of this present study is to evaluate Mg, VEGF, and OPN levels in healthy dogs and in dogs with mammary tumors with/without pulmonary metastases, and to investigate the alterations of these parameters in the serum and tissue samples of dogs with mammary tumors in connection with the histological tumor type and tumor grade. Mammary tumor groups were designed according to the presence of pulmonary metastasis in radiography; group MI consists of 20 dogs with metastatic mammary ttunors to the lung, and group MO consists of 20 dogs with nonmetastatic mammary tumors. Ten clinically healthy dogs composed group H. The dogs represented different breeds and ages. Three-view thoracic radiographs were taken to determine any metastasis in lungs by digital radiography. Magnesium, VEGF, and OPN were determined in the mammary gland samples and blood serum of 40 dogs with malignant mammary tumors and in 10 healthy dogs. The magnesium levels were measured by atomic absorption spectrophotometry, both in the tissue and serum samples. Also, the tissue and serum VEGF and OPN levels were determined by ELISA with commercially available kits. The tissue Mg levels in the MO group were significantly (P<0.05) higher than in group H. However, the serum VEGF level was significantly associated with a tumor type. Additionally, the serum OPN levels exhibited a tendency to be higher in dogs with mammary tumors with pulmonary metastases, grade 3, and eaminosarcoma. It is concluded that Mg, VEGF, and OPN could have practical use for diagnosing and understanding the pathophysiology of CMT.