Analysis of the presence of osteocalcin, S-100 protein, and proliferating cell nuclear antigen in cells of various types of osteosarcomas. Osteosarcomas are characterized by different histologic subtypes that are composed of heterogeneous tumor cells. Although the histological origin of the malignant cells is unknown, it has been speculated that osteoblasts lead to the malignant cells. In the current study, the osteosarcoma cells in 27 lesions were assessed by means of immunohistochemical staining for osteocalcin (OC), S-100 protein (S-100) and proliferating cell nuclear antigen (PCNA). PCNA labeling indices were the highest in osteoblastic and stromal areas, and significantly lower in chondroblastic areas (p < 0.01). Cells that were positive for both PCNA and OC were abundant in osteoblastic and stromal areas, while cells that were positive for both PCNA and S-100 were rarely observed. These results were almost similar for conventional, parosteal and periosteal osteosarcomas. In contrast, OC reactivity was poor in fibroblastic osteosarcoma, in osteosarcoma with giant cells, and in telangiectatic osteosarcoma. Pulmonary metastatic osteosarcoma lesions weakly expressed OC (p < 0.01), but showed high values for the PCNA labeling indices. In conclusion, immunohistochemical staining for OC, S-100, and PCNA are useful to analyze the proliferating cells in osteosarcomas. The main proliferating cells in most osteosarcomas are mature osteoblast-like cells. OC-negative tumor cells predominate in some of osteosarcoma subtypes, and these tumors therefore probably represent a distinct osteosarcoma variant. OC expression in pulmonary metastatic lesions may be suppressed.