DiGeorge syndrome (DGS) is a developmental defect characterized by cardiac defects, facial dysmorphism, and mental retardation. Several studies have described a critical region for DGS at 22q11, within which the majority of DGS patients have deletions. We have isolated nine cosmid and three YAC clones using previously described and newly isolated probes that have been shown to be deleted in many DGS patients. Using fluorescence in situ hybridization and digital imaging, we have mapped and ordered these clones relative to the breakpoints of two balanced translocations at 22q11 (one in a DGS patient and one in the unaffected parent of a DGS child). Our data indicate that the breakpoint in the unaffected individual distally limits the DGS critical region (defined as the smallest region of overlap), while proximally the region is limited by repeat-rich DNA. The critical region includes the balanced translocation breakpoint of the DGS patient that presumably disrupts the gene causing this syndrome.