A replication defective herpes simplex virus-1 was evaluated as a therapeutic vector. Mantle cell lymphoma (MCL), hairy cell leukemia (HCL), and B-cell chronic lymphocytic leukemia (B-CLL) were chosen because leukemic cells were collectable from peripheral bloods in these diseases. Cells from six MCL, one HCL, and nine B-CLL were infected in vitro with T0Z.1 at 3 multiplicity of infection (MOI). Herpes simplex virus thymidine kinase (HSV-TK)/ganciclovir (GCV)-mediated suicide gene therapy showed 14.7% of mean tumor killing against leukemic B-cell malignancies. The mean tumor-killing effects were 8.7 and 17.1% in MCL and B-CLL, respectively. The effect against HCL was 29%. The study indicates that herpes simplex virus (HSV)-based gene therapy might be an effective strategy.