Although IL-6 has been identified as a major growth factor in multiple myeloma (MM), it is believed that maintenance of tumor growth in vivo depends on one or more additional stroma-derived factors. We describe a new human myeloma cell line (MM5.1) that can be maintained in the presence of bone marrow-derived stromal cell layers, and not only when cultured with exogeneous IL-6. This cell line expresses the same immunoglobulin kappa light chain RNA sequence as the patient's original tumor cells, has a plasma cell morphology and expresses plasma cell antigens (cytoplasmic kappa light chain, CD38, BB4). Without the presence of stromal factors, MM5.1 cells become apoptotic. A low proliferative effect was observed in the presence of oncostatin M (OSM) but other cytokines (IL-10, IL-11, stem cell factor (SCF) and leukemia inhibitory factor (LIF)) had no effect at all. We observed that MM5.1 cells also grow when physically separated from stromal cell layers by a 0.45 microm microporous membrane or when cultured in conditioned medium from stromal marrow cells. Unexpectedly, the growth in stromal supernatants was markedly inhibited by an anti-IL-6 antiserum and an anti-IL-6 receptor transducer chain (gp130) mAb in a dose-dependent manner. This implies that MM5.1 cells are IL-6 responsive only when exposed to one or more additional soluble factor(s) derived from bone marrow stroma. Coculturing MM5.1 cells with IL-6 and cytokines that were described to increase the IL-6 responsiveness of myeloma cells (G-CSF, GM-CSF and IL-3) had no effect on the growth or survival. A strong proliferative effect was observed when MM5.1 cells were cultured with IL-6 and soluble IL-6 receptor (sgp80). However no sgp80 could be detected in stromal supernatants using a sensitive immunoassay. This indicates that sustained proliferation of the MM5.1 cell line depends on a combination of IL6 and at least one, thus far unidentified, stroma-derived factor. After more than 1 year in continuous culture, we could obtain a variant of the line (MM5.2) that shows an improved growth rate and grows stroma independently. Molecular analysis revealed clonal identity with the early passage form and Epstein-Barr virus antigen expression was negative. The two variants of this cell line offer a useful model to identify molecular mechanisms involved in clonal evolution towards stroma-independent growth of myeloma cells.