Glioblastoma multiforme, the most malignant human brain tumor, may develop de novo (primary glioblastoma) or through progression from low-grade or anaplastic astrocytoma (secondary glioblastoma). We present further evidence that primary and secondary glioblastomas constitute distinct disease entities which develop through the acquisition of different genetic alterations. We analyzed p53 mutations, p53 protein accumulation and epidermal growth factor receptor (EGFR) overexpression in 49 biopsies classified as primary or secondary glioblastoma according to clinical and histopathologic criteria. Patients with primary glioblastoma were selected on the basis of a clinical history of less than 3 months and histopathologic features of glioblastoma at the first biopsy (19 cases; mean age, 55 years). The diagnosis of secondary glioblastomas required at least two biopsies and clinical as well as histologic evidence of progression from low grade or anaplastic astrocytoma (30 cases; mean age, 39 years). DNA sequence analysis showed that p53 mutations were rare in primary glioblastomas (11%) while secondary glioblastomas had a high incidence of p53 mutations (67%), of which 90% were already present in the first biopsy. The incidence of p53 protein accumulation (nuclear immunoreactivity to PAb 1801) was also lower in primary (37%) than in secondary glioblastomas (97%). In contrast, immunoreactivity for the EGF receptor prevailed in primary glioblastomas (63%) but was rare in secondary glioblastomas (10%). Only one out of 49 glioblastomas showed EGFR overexpression and a p53 mutation. These data indicate that overexpression of the EGF receptor and mutations of the p53 tumor suppressor gene are mutually exclusive events defining two different genetic pathways in the evolution of glioblastoma as the common phenotypic endpoint.