Osteosarcomas are malignant bone tumors consisting of cells with abnormal cellular functions. Although osteosarcoma-derived cells are commonly used for osteoblastic models, the molecular composition of the osteosarcoma extracellular matrix (ECM) is not well characterized. We compared three osteosarcoma cell lines (MG-63, Saos-2 and U-2 OS) with normal human osteoblasts by immunocytochemistry. Cellular characteristics were assessed by morphometric analysis and proliferation kinetics. All investigated osteosarcoma cell lines exhibited very heterogeneous labelling profiles and each differed significantly from that of normal osteoblasts. Saos-2 cells revealed the most mature osteoblastic labelling profile while U-2 OS cells were negative for most of the investigated osteoblastic markers. We conclude that each osteosarcoma cell line exhibits a characteristic labelling profile and thus produces a differently composed extracellular matrix. This can be used in attempts to better characterize osteosarcoma, a as well as for their diagnosis.