OBJECTIVE: Multifocal glioblastomas constitute an increasingly diagnosed subgroup of glioblastoma multiforme, the most malignant primary brain tumor in adults. The molecular background of these lesions is unknown. However, the ability to study multiple lesions of one patient simultaneously could provide new aspects in glioma development. METHODS: Short-term cell cultures were derived from three isolated glioblastoma lesions of one patient. Spectral karyotyping and interphase fluorescence in situ hybridization were used for cytogenetic analysis. Loss of heterozygosity was assessed in tumor tissues and cell lines for seven gene loci (p73, p21, p16, PTEN, p27, Rb, p53). In addition, sequence analysis of the PTEN and p53 loci was performed, epidermal growth factor receptor protein expression was assessed, and in vitro proliferation was assayed. RESULTS: A balanced translocation [t(1;15)(p3?6;q2?5)] that has not been described previously in glioblastomas was identified in all cell lines. Primarily, the cell lines have a homozygous deletion of the p16 locus and inactivation of the PTEN gene by loss of heterozygosity and an identical mutation in common. Furthermore, the cell lines harbor a hemizygous (R175H) or two heterozygous (R175H, R213Q) mutations of the p53 gene or none at all. The occurrence of p53 mutations correlates with the size of the original tumors and in vitro proliferation. CONCLUSION: The analysis of a multifocal glioma revealed three main aspects: 1) the combined cytogenetic and molecular analysis of this subgroup of glioblastoma multiforme is a suitable tool to gain new perspectives in glioma development, 2) the balanced translocation [t(1;15)(p3?6;q2?5)] might harbor a new genetic marker involved in glioma development, and 3) the pattern of p53 mutation suggests a role of p53 in the progression of malignancy, migration, and growth of this particular primary glioblastoma.