The polymorphism of amino acid residue 72 on the human p53 tumor-suppressor gene is a useful marker for detecting intragenic loss of heterozygosity (LOH). We examined the LOH of the p53 gene in human malignant astrocytomas by the polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) analysis using DNA extracted from frozen tissue sections under histologic examination. Eleven of 16 informative cases (69%) of the malignant astrocytomas were found to have LOH in the p53 gene. Sequential frozen sections were analyzed by immunohistochemistry using anti-p53 antibody PAb1801 to detect overexpression of the p53 protein, which is presumably altered if it is detectable. Ten of the 11 cases that had LOH of the p53 gene overexpressed the p53 protein. Moreover, 4 of the 11 patients with LOH of the p53 gene developed a second neoplasm in addition to an astrocytoma, possibly indicating genetic instability in these patients. These data suggest that alterations of the p53 gene may play an important role in the genesis of malignant astrocytoma. The combination of the PCR-RFLP method and immunohistochemical analysis using frozen tissue sections is a practical diagnostic tool for examination of human malignancies, including astrocytomas. astrocytomas.