Clonality studies of hematopoietic reconstitution after remission were performed in 24 female patients (pts) with leukemias characterized by specific molecular markers. At diagnosis, 13 pts had promyelocytic leukemia (PML) retinoic acid receptor-alpha (RAR-alpha)-rearranged acute promyelocytic leukemia (APL), 8 Philadelphia positive (Ph′+) break-point cluster region (BCR+) chronic myeloid leukemia (CML), and 3 Ph′+ (BCR+) acute lymphoblastic leukemia (ALL). All pts were analyzed at presentation and after Southern blot suppression of specific rearrangements after various treatments, including conventional chemotherapy, autologous or allogeneic bone marrow transplant (BMT), all-trans retinoic acid, and alpha-2b interferon. DNA from BM samples collected at diagnosis and, during remission phases, were subjected to Southern blot analysis with the M27 beta probe to detect X chromosome methylation differences, and with BCR, in CML and ALL cases, or PML/RAR- a probes for gene rearrangements, in APL cases. Twenty-one of the 24 pts had polyclonal methylation patterns at remission, together with disappearance of the specific rearrangement, whereas 3 pts retained the same single unmethylated DXS255 allele detected at diagnosis despite no evidence of gene rearrangement. Concerning these 3 pts, such an apparently clonal pattern was also observed in one case in T lymphocytes and skin-derived DNA; in a second case in BM fibroblasts and T lymphocytes; and, in the third case, in blood mononuclear cells obtained from her healthy female BM donor. All these 3 pts are in unmaintained clinical and cytogenetic remission after more than 20 months off therapy. These data suggest that (1) polyclonal and presumably normal hematopoiesis occurs in APL, CML, and Ph′+ ALL pts once the major burden of leukemic cells carrying a specific rearrangement is suppressed by treatment; and (2) unbalanced X chromosome methylation patterns, or aberrant methylation of X chromosome regions may be observed in some cases, most likely reflecting constitutional features simulating a clonal picture.