Restriction fragment length polymorphisms (RFLPs) of the X-chromosome genes hypoxanthine phosphoribosyl transferase (HPRT) and phosphoglycerate kinase (PGK) were studied in 34 female patients with primary myelodysplastic syndromes (MDS). Twelve patients (35%) were heterozygous at the HPRT or PGK loci for BamHI or BglI RFLPs, respectively. In eight patients showing PGK polymorphisms, clonality was determined by X-chromosome inactivation analysis. These included patients from different morphologic subtypes: four with refractory anemia (RA), two with RA and ring sideroblasts (RARS), one patient with RA with excess of blasts (RAEB), and one with chronic myelomonocytic leukemia (CMML). A monoclonal pattern of X-chromosome inactivation was observed in seven cases. In a further case characterized by bone marrow hypoplasia, peripheral blood (PB) leukocytes were polyclonal in origin. Following low-dose cytarabine therapy, reversion to polyclonal hematopoiesis was observed in a case of RAEB indicating the presence of residual normal hematopoietic stem cells with the capacity for marrow reconstitution. The clonal relation of lymphoid and granulocyte/monocyte lineages was studied directly in two cases of CMML exhibiting somatic mutations of N-ras or Ki-ras oncogenes. By selective oligonucleotide hybridization to ras gene sequences amplified in vitro by the polymerase chain reaction, a mutated ras allele was demonstrated in PB granulocytes, monocytes, and B and T lymphocytes of both patients. We conclude that MDS arise from a multipotent hematopoietic stem cell with the potential for myeloid and lymphoid differentiation.