Analysis of mutant P53 protein in osteosarcomas and other malignant and benign lesions of bone. Alterations of tumour suppressor genes are considered crucial steps in the development of human cancers. Expressions of p53 protein, a product of the tumour suppressor gene altered most commonly in human cancers examined so far, were investigated immunohistochemically in 18 osteosarcomas and 40 other malignant and benign lesions of bone. A monoclonal antibody clone PAb240, which recognizes a common conformational epitope of mutant p53 proteins, stained nuclei of tumour cells in 12 of 18 osteosarcomas (67%). Six tumours (33%) particularly showed positive immunoreactions in more than half of the tumour cells. PAb240 also stained tumour cells in a small number of other malignant bone tumours, such as malignant fibrous histiocytoma, chondrosarcoma, and Ewing's sarcomas. Furthermore, a small number of cells of giant-cell tumours were positively stained. In contrast, PAb240 was completely negative in 21 benign bone tumours and reactive lesions examined. Another monoclonal antibody clone PAb1801, which reacts with both wild- and mutant-type p53 protein, reacted in nuclei of tumour cells of 7 osteosarcomas (39%). Most of those also reacted with PAb240. PAb1801 was expressed much more frequently in other malignant bone tumours and giant-cell tumours. In addition, PAb1801 showed intranuclear positive reactions in tumour cells of a benign chondroblastoma, and reactive cells such as actively proliferating preosteoblasts in a myositis ossificans and osteoclast-like giant cells in a giant-cell tumour. The immunoelectron-microscopic observation that p53 protein was localized in euchromatic areas of nuclei of osteosarcoma cells supported the specificity of immunoreaction for p53 protein, indicating an active role of p53 protein in the regulation of DNA synthesis and transcription. These findings suggest that point mutation of the p53 gene is frequently involved in the development of osteosarcomas. PAb240 may be a useful tool not only in screening point mutations of the p53 gene in osteosarcomas but also in the differential diagnosis between osteosarcomas and reactive bone-forming lesions. Expressions of mutant p53 protein were not correlated with any clinical or pathological factors examined, although the results should be confirmed in studies of a large number of osteosarcomas.