The Prader-Willi chromosome region (PWCR) in Prader-Willi syndrome patients was analyzed by using genomic DNA probes mapping to 15q11.2-q12. The present report includes analysis of dosage by RFLP and densitometric studies, and analysis of restriction patterns. Twelve Prader-Willi syndrome (PWS) patients were studied: 5 had deletions of 15q11-q13, one had an unbalanced translocation, and 6 were karyotypically normal. Four genomic DNA probes were used: pML34 (D15S9); pTD3-21 (D15S10); IR4-3 (D15S11), a subclone of IR4; and IR10-1 (D15S12), a subclone of IR10 (Donolon et al.: Proc Natl Acad Sci USA 83:4408-4412, 1986). The results presented demonstrate that molecular rearrangements have occurred in 10 of the 12 PWS patients investigated and that the specific rearrangements differ from patient to patient. Patients with apparently similar cytogenetic deletions differ at the molecular level with deletions and/or duplications of various loci. The present study reports molecular alterations within the PWCR in PWS patients reported as cytogenetically normal. However, the 6 karyotypically normal patients are a heterogeneous group with molecular rearrangements ranging from none detected to deletions and/or duplications. These molecular studies suggest that a physical disruption of the PWCR causes the PWS not only in those patients reported to have a cytogenetic aberration but also in those identified as apparently karyotypically normal. The question remains as to whether the PWS patients in whom a molecular abnormality has not been detected have an autosomal recessive form of PWS, a molecular disruption which has not yet been detected, or another mechanism producing an apparently identical phenotype. The order of the 4 loci on chromosome 15 is hypothesized to be cen----D15S9----D15S12----D15S11----D15S10.