We studied CD81 expression by flow cytometry (FC) on benign precursor B cells (hematogones) and leukemic blasts in precursor B-cell acute lymphoblastic leukemia (pre-B-ALL) and established its usefulness in minimal residual disease (MRD) assays. Hematogones showed uniformly bright CD81 expression. In 98 pre-B-ALLs at diagnosis or overt relapse, 80 (82%) showed aberrantly decreased CD81 intensity. We used hematogones in 139 MRD- specimens to set a lower threshold for normal CD81 expression. In 133 specimens positive for residual pre-B-ALL, 87.2% showed increased CD81-dim immature B cells (>10%) and/or a discrete cluster of CD81-dim cells in a background of hematogones. Only 1 of 139 MRD- specimens showed more than 10% CD81-dim cells. Decreased CD81 expression was maintained in 91% of aberrant cases analyzed before and after chemotherapy. Decreased CD81 expression is a sensitive and specific marker for residual pre-B-ALL, even in a background of hematogones, making CD81 a useful addition to a panel for MRD detection by FC.