The clinical features of the 9p-deletion syndrome include dysmorphic facial features (trigonocephaly, midface hypoplasia, upward-slanting palpebral fissures, and a long philtrum) and mental retardation. The majority of these patients appear to have similar cytogenetic breakpoints in 9p22, but some cases show phenotypic heterogeneity. To define the breakpoints of the deleted chromosomes, we studied 24 patients with a deletion of 9p, by high-resolution cytogenetics, FISH with 19 YACs, and PCR using 25 different sequence-tagged sites. Of 10 different breakpoints identified, 9 were localized within an approximately 5-Mb region, in 9p22-p23, that encompasses the interval between D9S1869 (telomeric) and D9S162 (centromeric). Eight unrelated patients had a breakpoint (group 1) in the same interval, between D9S274 (948h1) and D9S285 (767f2), suggesting a chromosome-breakage hotspot. Among 12 patients, seven different breakpoints (groups 3-9) were localized to a 2-Mb genomic region between D9S1709 and D9S162, which identified a breakpoint-cluster region. The critical region for the 9p-deletion syndrome maps to a 4-6-Mb region in 9p22-p23. The results from this study have provided insight into both the heterogeneous nature of the breakage in this deletion syndrome and the resultant phenotype-karyotype correlations.