Herpes simplex virus (HSV)-based vectors have favorable biologic features for gene therapy of leukemia and lymphoma. These include high transduction efficiency, ability to infect postmitotic cells, and large packaging capacity. The usefulness of HSV amplicon vectors for the transduction of primary human B-cell chronic lymphocytic leukemia (CLL) was explored. Vectors were constructed encoding beta-galactosidase (LacZ), CD80 (B7.1), or CD154 (CD40L) and were packaged using either a standard helper virus (HSVlac, HSVB7.1, and HSVCD40L) or a helper virus-free method (hf-HSVlac, hf-HSVB7.1, and hf-HSVCD40L). Both helper-containing and helper-free vector stocks were studied for their ability to transduce CLL cells, up-regulate costimulatory molecules, stimulate allogeneic T-cell proliferation in a mixed lymphocyte tumor reaction, and generate autologous cytotoxic T lymphocytes (CTLs). Although helper-containing and helper-free amplicon stocks were equivalent in their ability to transduce CLL cells, a vigorous T-cell proliferative response was obtained using cells transduced with hf-HSVB7.1 but not with HSVB7.1. CLL cells transduced with either HSVCD40L or hf-HSVCD40L were compared for their ability to up-regulate resident B7.1 and to function as T-cell stimulators. Significantly enhanced B7.1 expression in response to CD40L was observed using hf-HSVCD40L but not with HSVCD40L. CLL cells transduced with hf-HSVCD40L were also more effective at stimulating T-cell proliferation than those transduced with HSVCD40L stocks and were successful in stimulating autologous CTL activity. It is concluded that HSV amplicons are efficient vectors for gene therapy of hematologic malignancies and that helper virus-free HSV amplicon preparations are better suited for immunotherapy.