Abstract. Aspiration of lytic bone lesions is an excellent diagnostic test in the initial evaluation of primary bone neoplasia. However, cytologically, it can be difficult to differentiate osteosarcoma (OSA) from other bone neoplasms, including fibrosarcoma, chondrosarcoma, synovial cell sarcoma, and plasma cell myeloma. The purpose of this study is to determine the sensitivity and specificity of alkaline phosphatase (ALP) staining to differentiate OSA from other tumors that express vimentin by immunocytochemistry or immunohistochemistry. ALP is a hydrolytic enzyme present in multiple tissues including liver, kidney, intestine, placenta, and bone. Hypothetically, neoplasms actively producing bone should be specifically positive for ALP staining. Unstained, cytologic specimens were incubated for 8-10 minutes with nitroblue tetrazolium chloride/5-bromo-4-chloro-3-indolyl phosphate toluidine salt-phosphatase substrate. A positive reaction stains the membrane of the cells gray to black. Samples were counterstained with a Romanowsky's stain to determine whether the sample was of representative cellularity. A total of 61 vimentin-positive neoplasms have been evaluated and confirmed histopathologically. Tumors that expressed vimentin and were positive for ALP included 33 OSAs, one multilobular tumor of bone, one amelanotic melanoma, and one chondrosarcoma. Tumors that expressed vimentin and were negative for ALP included chondrosarcomas (three of four), multiple fibrosarcomas, and multiple synovial cell sarcomas. The sensitivity is 100%, and the specificity is 89%. In conclusion, ALP appears to be a highly sensitive and fairly specific marker in the diagnosis of OSA.Key words: Alkaline phosphatase; bone; cytology; osteosarcoma.Osteosarcoma (OSA) is the most common primary bone tumor of dogs, accounting for 85% of reported skeletal malignancies. 4,14 Biologically, the tumor is locally aggressive, with a high metastatic rate. 16 The majority of OSAs are appendicular, 75% of which originate from the distal radius or proximal humerus. 7 Histopathologically, OSA is described as a malignant spindle cell tumor characterized by the production of an osteoid matrix by tumor cells. 16 The gold standard for diagnosis is considered biopsy with histopathologic evaluation. 7 Bone biopsy is an invasive procedure, often with a delayed diagnosis because of the decalcification process. In addition, bone biopsies can have complications, such as an increased risk of pathologic fracture at the biopsy site. Fine needle aspiration of lytic bone lesions is becoming more common in human medicine. 1,9,18 In one study, fine needle aspiration and cytology of bone tumors revealed a sensitivity of 86% and a specificity of 94.7%, with histopathology as the gold standard. 1 In veterinary medicine, aspiration of lytic lesions is gradually being used more frequently. In a recent study by Cohen et al., the overall sensitivity and specificity of cytology was evaluated. Although this study was not limited to bone, nine bone aspirations were evaluated, with...