B lymphocytes are attractive targets for gene therapy of genetic diseases associated with B-cell dysfunction and for immunotherapy. Transduction of B lymphocytes was evaluated using green fluorescent protein (GFP)-encoding onco-retroviral and HIV-derived lentiviral vectors which were pseudotyped with ecotropic, amphotropic or vesicular stomatitis virus (VSV-G) envelopes. Transduction of mouse B lymphocytes activated with lipopolysaccharides (LPS) or by cross-linking CD40 in conjunction with interleukin-4 (IL-4) was significantly more efficient (p < 0.003) with ecotropic (11%) than with VSV-G pseudotyped onco-retroviral vectors (1%). Using high-titer cell-free ecotropic viral supernatant or by coculture with ecotropic onco-retroviral vector-producing cells, transduction efficiency increased significantly (p < 0.001) to approximately 50%, whereas transduction efficiency by coculture with VSV-G pseudotyped vector-producing cells remained low (< 2%). Similarly, transduction of mouse B lymphocytes was significantly more efficient (twofold, p < 0.01) with the ecotropic (7%) than with the VSV-G pseudotyped lentiviral vectors although gene transfer efficiency remained low because of dose-limiting toxicity of the concentrated vector preparations on the LPS-activated murine B cells. Consistent with murine B-cell transduction, human B cells activated with CD40L and IL-4 were also found to be relatively refractory to VSV-G pseudotyped onco-retroviral vectors (< 1%). However, higher transduction efficiencies could be achieved in activated primary human B lymphocytes using VSV-G pseudotyped lentiviral vectors instead (5%-6%). Contrary to the significant increase in mouse B-cell transduction efficiency with ecotropic vectors, the use of amphotropic onco-retroviral or lentiviral vectors did not increase transduction efficiency in primary human B cells. The present study shows that the transduction efficiency of onco-retroviral and lentiviral vectors in human and mouse B lymphocytes is pseudotype-dependent and challenges the widely held assumption that VSV-G pseudotyping facilitates gene transfer into all cell types.