Total cell production and longevity of hematopoiesis in long-term bone marrow culture of tumor necrosis factor (TNF)-deficient mice (LTBM-TNFko) are increased. The rate of apoptosis is decreased during the first 40 weeks in culture, then the level of apoptosis reaches levels of wild-type cultures. Extended lifespan of primary cultures usually is the consequence of the neoplastic transformation. We set out to check this possibility in the LTBM-TNFko. Telomerase activity in suspension fraction (SF) of LTBM-TNFko increases with time and reaches maximum a year after culture initiation. Cytogenetic study reveals genome instability in SF and hyperploidy in the adhesion cell layer (ACL) of LTBM-TNFko. All of the above indicate the possibility of neoplastic transformation. However, histological study of cells and CFU-S-derived colonies of SF does not reveal a block of differentiation. Cells of SF are unable to grow without ACL. Although those cells could proliferate in the presence of exogenous growth factors, they are not able to be passaged. Attempts of passaging ACL cells failed as well. Neither healthy nor sublethally irradiated recipients injected intravenously or intraperitoneally with cells of SF develop tumors within 8 months of observation. In conclusion, abnormal dynamics of long-term bone marrow culture of TNF-deficient mice could not be explained by neoplastic transformation.