We have obtained lymphoblastoid cell lines from three patients with Langer-Giedion syndrome who have overlapping deletions in 8q24.1. To isolate the deletion chromosomes from their normal homologs, patient cell lines were fused with hamster cells and hybrid cells were selected for retention of human chromosome 8. These hybrid cell lines were screened for the presence of chromosome 8 by fluorescence in situ hybridization and by Southern blot hybridization. We have hybridized 31 recombinant DNA clones derived from the 8q22-qter region to Southern blots of the hybrid cell lines; 8 were found to lie within the deletion of at least one patient. One clone identified sequences that were missing from one copy of chromosome 8 in all three patients. These clones help to further define the deletions in these patients and will serve as starting points for detailed characterization of the region.