The expression of RNA transcripts from two pre B lymphocyte related genes, VpreB and λ5, has been studied in a series of transformed cell lines which appear frozen at different states of B lineage differentiation, from early progenitors to surface Ig positive B cells. In the HAFTL-1 cell line, which arose from fetal liver by transformation with a retrovlrus containing the Hras oncogene, Northern analysis of poly A+ mRNA as well as in situ hybridization of RNA In single cells revealed that λ5 and VpreB are already expressed at the progenitor stage and increase in expression as the progenitors differentiate to precursor (preB) cells, or are turned off as the progenitors differentiate to myeloid cells. Continued rearrangements of Ig genes in pre B cell lines leading to Ig expression on the surface of NFS-5 pre B cells do not influence the continued expression of VpreB and λ5. Surface Ig-positive B lineage cell lines also express the pre B-related genes. Both Ly1+ as well as Ly1- pre B cells are VpreB and λ5positlve. Lipopolysaccharide (LPS) stimulation of 70Z/3 pre B cells does not turn off λ5 expression. It therefore appears that, at least In transformed cell lines, the expression of VpreB and λ5, is not directly regulated by the expression of μH, κL, or λL chains, LPS reactivity, or the Ly1 surface antigen. Fusion of plasmacytoma cells with normal pre B cells to generate pre B hybridomas leads to down-regulation of VpreB/λ5 expression. These results suggest that different trans-acting factors in more mature cells might down-regulate the expression of VpreB/λ5 © 1992 Oxford University Press.