Rearrangements of EGFR are known to occur in a significant fraction of glioblastomas, the most common and malignant form of central nervous system tumor. Although the consequences of these alterations have been described at the mRNA and protein level, little is known about human EGFR genomic sequence or organization at the rearrangement sites. To investigate one group of alterations in glioblastoma, we used long-range PCR to synthesize a segment of the gene near its 3' end, which is frequently rearranged in tumors with EGFR amplification. The sequence of this PCR product provided a precise map for the five 3'-terminal exons of EGFR, designated as exons 22-26. Ten tumors were identified with rearrangements in this part of the gene, most of which resulted in the loss of 325 coding bases that constitute exons 23-25. No two tumors shared identical donor or acceptor rearrangement sites, and the examination of sequences at these sites failed to support homologous recombination as a mechanism responsible for any of the rearrangements. However, examination of the entire exon 22-26 region for sequence motifs associated with genomic instability identified two large, CA-rich tracts in intron 25.