CD40 ligand (CD40L) is a good candidate molecule for the immunotherapy of B cell malignancies including B-chronic lymphocytic leukemia (B-CLL), because it may increase the capacity of the malignant cells to present tumor antigens. However, efforts to manipulate expression of the human CD40L (hCD40L) molecule have foundered on problems associated with lack of consistent gene transfer into the malignant target cells. We now describe a new, highly reproducible method for inducing hCD40L surface expression on malignant B cells, which is dependent on intercellular transfer of the hCD40L protein from donor gene-modified fibroblasts to patient tumor cells. Ten B-CLL samples were cocultured with MRC-5 fibroblasts (a human embryonic lung cell line) previously transduced with an adenoviral vector encoding the hCD40L gene. The malignant cells expressed high levels of surface hCD40L, B7-1, B7-2, and ICAM-1 after coculture. Upregulation of B7-1 and B7-2 was cycloheximide inhibitable and was a consequence of CD40 activation. However, inhibition of protein synthesis had no effect on the ability of B-CLL cells to acquire surface expression of hCD40L. hCD40L surface expression required cell-to-cell contact, but was independent of CD40 engagement. hCD40L transfer was not mediated by membrane fusion. The transferred hCD40L was functionally intact and B-CLL cells expressing this molecule induced increased interferon-gamma production from autologous peripheral blood T lymphocytes. This approach does not use any direct gene transfer to primary leukemia cells and can readily be scaled up for production of clinical B-CLL vaccines.