Long-term culture of a nonadherent pre-B cell line was established from BALB/c bone marrow by the method of Whitlock and Witte. Adherent filler cells, also derived from bone marrow, were required for the growth of such pre-B cells. The surface phenotypes of the nonadherent cells determined by FACS analysis were negative for mu-chain, kappa-chain, and Ia, but 50% of nonadherent cells expressed a marker recognized by the monoclonal 14.8. Analysis of cytoplasmic immunofluorescent staining revealed the presence of cytoplasmic mu-chain but absence of kappa-chain. These pre-B cells did not respond in vitro to such mitogens as LPS or DxSO4 and were not stimulated by allospecific helper T cells. However, when such BALB/c derived pre-B cells were transferred to irradiated BDF1 mice, recovered spleen cells expressed mu-chain as well as the marker recognized by 14.8. Such in vivo passaged pre-B cells were able to respond to LPS in vitro. Moreover, the response observed was diminished markedly by anti-H-2d antiserum and complement (C) but not anti-H-2b and C, indicating that the responder cells were of donor rather than host origin. Because the reconstitution of irradiated mice was limited to cells of the B lymphocyte lineage, it would appear that cells recovered from in vivo passage are the progeny of in vitro cultured pre-B cells and not that of in vitro contaminating stem cells.