A number of epidermal growth factor receptor (EGFR) deletion mutants have been identified in gliomas, in which the EGFR gene is frequently amplified and rearranged. We have previously characterized the structure of a gene in A-172 human glioma cells that encodes a 190-kDa EGFR mutant with tandem duplication of the tyrosine kinase (TK) and calcium-mediated internalization (CAIN) domains. Here we describe a 185-kDa tandem duplication mutant (TDM) that is expressed in KE and A-1235 glioma cells, along with certain functional characteristics of the mutants. The corresponding transcripts in KE and A-1235 cells contain 1053 additional nucleotides representing an in-frame duplication of exons 18 through 25 which encode the entire TK region and a portion of the CAIN domain. As with duplication of the entire TK/CAIN region (exons 18-26) in A-172 cells, duplication of exons 18-25 is associated with a specific genomic rearrangement between flanking introns. Involved introns contain homology to recombination signal sequence (RSS) heptamers present in the V(D)J region of the T lymphocyte receptor gene. In defined medium, both oncogenic TDM are constitutively autophosphorylated and inefficiently downregulated. High-affinity binding is reduced in EGFR.TDM/18-26, although the t1/2 of receptor internalization is not prolonged.