The feasibility of sequential positive and negative selection of mobilized CD34+ B-lineage negative cells to achieve tumour-free autografts in multiple myeloma (MM) patients was evaluated. Peripheral blood stem cells (PBSC) of 14 patients with advanced disease were mobilized. CD34+ cells were enriched in 12 of the patients by the avidin-biotin immunoabsorption technique. Subsequently, CD10+, CD19+, CD20+ and CD56+ cells (B-lin cells) were removed by immunomagnetic depletion. Minimal residual disease (MRD) was detected by flow cytometry and PCR-based molecular analysis of the patient specific IgH complementary-determining region III (CDRIII). Positive selection of stem cells produced a median recovery of 54.7% of the initial content of CD34+ cells (median purity 71.9%). Negative depletion of B-lineage cells reduced the number of CD34+ cells to 33.3% of the baseline value (median purity 72.7%). However, long-term culture assays showed the recovery of >60% of primitive haemopoietic progenitor cells after depletion of the B-lineage-positive cells. All evaluable patients had detectable disease in PBSC collections. The first step of positive selection of CD34+ cells resulted in >2 logs of tumour cell purging. However, molecular assessment showed the persistence of the disease in 6/7 cases. Immunofluorescence analysis demonstrated 1 additional log of B-cell purging by negative depletion. More importantly, molecular evaluation of IgH CDRIII region showed the disappearance of myeloma cells in 6/7 patients. 12 patients received a median of 3.9 x 106 CD34+ B-lin- cells/kg after conditioning with high-dose melphalan and showed a rapid reconstitution of haemopoiesis. These results were similar to two similar cohorts of patients who received either unmanipulated PBSC or positively selected CD34+ cells after the same conditioning regimen. Severe extrahaematological toxicity was limited to mucositis; no late infections were observed. We concluded that autotransplantation of purified CD34+ B-lin- cells was associated with a rapid and sustained recovery of haemopoiesis and low peritransplant morbidity. Sequential positive and negative enrichment of stem cells reduced tumour cell contamination in B-cell malignancies below the lower limit of detection of molecular analysis.