Leukemic cells frequently persist in the bone marrow of patients treated for acute lymphoblastic leukemia (ALL), and may regrow to produce relapse. We have used a long-term co-culture system to analyze the interaction of ALL blasts with components of the marrow microenvironment. Blast cells from 10 cases of precursor-B ALL were cultured on allogeneic human bone marrow stromal layers at 37 degrees C in microtiter wells, and replated when stroma showed evidence of deterioration. Leukemic cells from seven of 10 cases showed evidence of survival and proliferation beyond 30 days in culture, while in three cases there was a progressive decline in the number of viable leukemic cells by 7-21 days. Two cases continued to proliferate for 149 to 332+ days, while five underwent senescence after 34-52 days. In the two cases with long-term proliferation, the leukemic identity of the cells was confirmed by immunophenotyping, cytogenetics, and demonstration of clonal immunoglobulin gene rearrangements. Evidence of selection of leukemic subclones was seen in two cases, with immumophenotypic evidence of loss of CD10 and CD34 antigens, and acquisition of CD20 and surface mu chain. The leukemic cells in these cases grew either in clumps attached to the surface of the stroma, or as'cobblestone areas' beneath the stromal cells. Survival and growth of two evaluable cases was dependent on the continuing presence of stromal cells in the culture system. In one case, direct contact with stroma was shown to be necessary to main- tain viability, while blast cells from the other case survived equally well when separated from the stroma by a 0.4-micron pore size microporous membrane. These results indicate that leu- kemic cells from the majority of cases of precursor-B ALL are able to persist and undergo proliferation in vitro in the presence of normal marrow stroma. This process appears dependent on either direct cell-cell contact, or on diffusible factors derived from the stroma. The availability of ALL cells capable of indefinite proliferation under these conditions will allow further analysis of the mechanisms mediating leukemic cell proliferation.