Serial bone marrow aspirates from patients previously given a diagnosis of acute lymphoblastic leukemia (ALL) who had undergone chemotherapy, bone marrow transplantation (BMT), or both were analyzed by multidimensional flow cytometry (MDF) to detect residual disease (lower limit of detection 0.3%). Correlation between the results of morphologic examination and MDF showed concordant results on 100 of 118 specimens. The MDF-positive, morphologic examination-negative specimens were positive by cytogenetic examination or were obtained from patients in whom the ALL eventually relapsed. Similar correlations between MDF and the results of cytogenetic examination were obtained. Leukemic cells were detected in 29 of 62 patients before BMT and 12 of 52 after BMT Normal regenerating lymphoblasts were identified and quantified by MDF in patients without detectable leukemic lymphoblasts. Patients with leukemic lymphoblasts found by MDF in specimens obtained immediately before BMT were 3.28 times more likely to experience relapse after BMT compared with MDF-negative patients, even when leukemic lymphoblasts were undetectable by histopathologic examination, cytogenetic examination, or both. All patients who had undergone BMT with leukemic lymphoblasts found by MDF, with or without morphologic or cytogenetic confirmation, experienced relapse according to conventional criteria within 42 days of the MDF analysis. The detection of residual disease before overt relapse may provide information for early intervention, while definitive recognition of normal recovering blasts may prevent unnecessary treatment.