To characterize some of the genetic events underlying the development of glioblastoma multiforme, the authors analyzed 65 astrocytic tumors (seven pilocytic astrocytomas, eight astrocytomas, 16 anaplastic astrocytomas, and 34 glioblastomas multiforme) for loss of heterozygosity for chromosome 17p, loss of heterozygosity for chromosomes 10p and 10q, amplification of the epidermal growth factor receptor (EGFR) gene, and amplification of the oncogenes N-myc, c-myc, and N-ras using Southern blot analysis. Alterations of the p53 gene (positive immunostaining for p53 protein in tumors with or without p53 gene mutations) in these 65 tumors were analyzed previously. None of the 65 tumors showed amplification or rearrangement of N-myc, c-myc, or N-ras oncogenes. The molecular analysis presented here demonstrates distinct variants of astrocytic tumors, with at least three genetic pathways leading to glioblastoma multiforme. One pathway was characterized by 43 astrocytomas with alterations in p53. Glioblastomas with p53 alterations may represent tumors that progress from lower-grade astrocytomas. This variant was more likely to show loss of chromosome 17p than tumors without p53 alterations (p < 0.04). Seventy-five percent of tumors with loss of one 17p allele demonstrated mutations in the p53 gene. Loss of chromosome 10 was associated with progression from anaplastic astrocytoma (13%) to glioblastoma (38%) (p < 0.04). Amplification of the EGFR gene was a rare (7%) but late event in tumor progression (p < 0.03). A second pathway was characterized by six astrocytomas without p53 alterations and may represent clinically de novo high-grade tumors. These tumors were more likely to show amplification of the EGFR gene (83%) than tumors with p53 alterations. Sixty percent of tumors with EGFR amplification also showed loss of chromosome 10; loss of chromosome 17p was infrequent in this variant. One or more alternative pathways were characterized by 16 astrocytomas without p53 alterations and with none of the genetic changes analyzed in this study. Glioblastomas are a heterogeneous group of tumors that may arise via multiple genetic pathways.