A major obstacle to successful autologous bone marrow transplantation in breast cancer is infiltration of the marrow by malignant cells. We layered bone marrow samples from seven breast cancer patients on a discontinuous bovine albumin gradient, then assessed hematopoietic potential (colony-forming units-culture [CFUc]) and clonogenic tumor cells (TCFUc) by standard techniques. We found that 78% of CFUc concentrated in fraction 3 of the gradient, which contained 10% of the total nucleated cell population. TCFUc were distributed across the gradient with 14% of colonies identified in this marrow fraction. We applied these techniques to two patients with metastatic breast cancer who were treated with high-dose mitomycin-C, doxorubicin, and cyclophosphamide before receiving 1.9 x 10(9) and 1.2 x 10(9) total cells, respectively, from CFUc-rich fraction 3. We observed tumor colony growth in three patients only in separated marrow fractions, suggesting that colony growth may be a function of the cell composition after fractionation or that growth factors may be separable. Ninety percent of clonogenic breast cancer cells can be separated from hematopoietic cells by discontinuous density gradient fractionation, a technique that is applicable to the large volumes necessary in bone marrow transplantation and that may be an important initial step in marrow purging for autologous transplantation.