The highly packed cell density and the three-dimensional structure in the hematopoietic compartment of bone marrow facilitate cell-to-cell and cell-to-matrix interactions known to be important for hematopoietic activities. To provide a similar environment in vitro, we developed a long-term bone marrow culture (LTBMC) system, continuously perfused with Dexter's medium, employing packed, highly porous bovine collagen microspheres as the matrix support for marrow cell growth. Using murine bone marrow as a model, we found that the culture system differed from the conventional flask culture in the following ways: 1) as revealed by the electron microscopy, the bone marrow cells in the culture system grew in a three-dimensional configuration, similar to that in vivo, 2) the cell output from the culture system at 37 degrees C was virtually the same as that at 33 degrees C, and 3) in the absence of exogenous growth factors, except those in the serum, the culture system produced lymphoid cells and all stages of committed cells (i.e., erythrocytes, granulocytes, macrophages, and megakaryocytes), thus indicating multilineal differentiation of the hematopoietic stem cells. Furthermore, cell clusters resembling erythroblastic islands were observed in the absence of exogenous erythropoietin (Epo). The culture system appears to provide a different microenvironment than that of the flask culture and may be used as an alternative model for hematopoiesis.