Three new neurofibromatosis type 1 (NFl) mutations have been detected and characterized. Pulsed-field gel and Southern blot analyses reveal the mutations lo be deletions of 190, 40, and 11 kb of DNA. The 11 kb deletion does not contain any of the previously characterized genes that lie between two NFl translocation breakpoints, but it does include a portion of a rodent/human conserved DNA sequence previously shown to span one of the translocation breakpoints. By screening cDNA libraries with the conserved sequence, we identified a number of cDNA clones from the translocation breakpoint region (TBR), one of which hybridizes to an~ 11 kb mRNA. The TBR gene crosses at least one of the chromosome 17 translocation breakpoints found in NFl patients. Furthermore, the newly characterized NFl deletions remove internal exons of the TBR gene. Although these mutations might act by compromising regulatory elements affecting some other gene, these findings strongly suggest that the TBR gene is the NFl gene.