No effective therapy is available for the majority of the 30-40% of children with acute lymphoblastic leukemia (ALL) who relapse. Since the morphologically undetectable, or occult, leukemia cells that persist during remission originate from the clone present at diagnosis, may also have both the capability to sustain the disease and to give rise to relapse, we are evaluating a method of identifying them. We have combined, for the first time, an ALL blast colony assay (BCA) and the polymerase chain reaction (PCR) to isolate residual leukemia cells in remission bone marrow aspirate specimens from eight patients with B-precursor ALL during early continuation therapy. We found colony-forming leukemia cells with in vitro self-renewal capability that survived chemotherapy for 15 months after diagnosis in all sequential specimens from these patients. To verify the leukemic nature of these cells their DNA was amplified by PCR and the product directly sequenced. In every case, the VHDJH sequence observed at diagnosis was found. None of the patients relapsed during this early phase of their treatment, consistent with the observation that patients with B-precursor ALL experience recurrence late in their course. Since it is possible that some of these persistent leukemia cells belong to the leukemia progenitor cell population that sustains the disease, the study of them could provide the means to determine the mechanisms of relapse.