Leukaemic cells from most cases of B-chronic lymphocytic leukaemia die rapidly by apoptosis in vitro unless they are cultured in the presence of interleukin-4 or interferon alpha or gamma. We now report prolonged survival of purified CLL cells cultured on bone marrow (BM) derived stromal cells in the absence of exogenous growth factors. In 10 cases of CLL examined 0-61% (mean 14.7%) of the cells were viable after 10 d culture in medium alone, whereas in the presence of BM stromal cells 10-102% (mean 47.0%) of cells were recovered alive (P < 0.005) in 7/10 cases of CLL, cells remained viable after 30 d of culture in BM stromal cells with cell recovery of 12-65%. These long-term cultured CLL cells were Epstein Barr virus negative, shown by the failure to detect the ENBA-2 and BZLF1 genes of EBV by PCR analysis. Identity between day 0 and day 30 CLL cells was demonstrated by sequence analysis of their clonal IgH CDR3 region. Adherence of CLL cells to BM stromal cell layers was critical for their protection from apoptosis. Separation of CLL cells from stroma by 0.45 micron culture filters resulted in loss of the protective effect of the stromal cells. Stromal cells were also able to protect CLL cells from hydrocortisone-induced apoptotic cell death. Our findings provide an in vitro system that can be used to analyse the growth requirements of CLL cells and their chemosensitivity in an in vitro environment that mimics the in vivo milieu.