We investigated genetic heterogeneity of astrocytic gliomas using p53 gene mutations as a marker. Different parts of morphologically heterogeneous astrocytic gliomas were microdissected, and direct DNA sequencing of p53 gene exons 5 through 8 was performed. Thirty-five glioma samples and tumor-adjacent normal-appearing brain tissue from 11 patients were analyzed. Sixteen different p53 gene mutations were found in 7 patients. We found that some tumors were devoid of p53 gene mutations, whereas other tumors carried 1 or often several (up to 3) different mutations. The mutations were present in grade II, III, and IV astrocytic glioma areas. Both severe functionally dead mutants and mutants with remaining transcriptional activity could be observed in the same tumor. We observed that morphologically different parts of a glioma could carry different or similar mutations in the p53 gene and could be either associated or not associated with the locus of heterozygosity at the mutant site. Coexistence of p53 gene mutations and the locus of heterozygosity was common, at least in astrocytomas grade III and in glioblastomas, and also occurred in astrocytoma grade II areas. These results support the notion that intratumoral heterogeneity in brain tumors originates from different molecular defects. Our results are of importance for a further understanding of the molecular mechanisms behind failure to treat glioma patients.