The herpesvirus saimiri (HVS) genome has the capacity to incorporate large amounts of heterologous DNA and can be maintained episomally in many different human cell types. To evaluate the efficacy of HVS-mediated gene transfer into human hemopoietic cells, we investigated the ability of an HVS-based construct, carrying the enhanced green fluorescent protein (EGFP) and neomycin resistance genes, to transduce a variety of human hemopoietic cell lines and primary CD34+ cells. As measured by flow cytometry, the numbers of EGFP+ cells at 2 days postinfection differed between various cell types ranging, from 1.3% for KG1 cells to 56.8% for THP-1 cells. In addition, the expression of EGFP in Jurkat cells was retained at >95% per round of cell division over a period of 6 weeks (comparable with Epstein-Barr virus-derived gene therapy systems). Although the virus was not specifically disabled, no lytic viral mRNAs could be detected in transduced Jurkat cells, and infectious virus could not be detected by sensitive virus recovery assay. We also describe a simple centrifugation method that increases the efficiency of transduction by >100% in some cases and may be generally applicable to other herpesvirus-based vectors for ex vivo gene delivery. Using this technique, we were able to demonstrate a tropism for CD34+/CD14+ cells, transducing 30% of the population. These cells are known to give rise to dendritic cells (the most potent of the antigen-presenting cells), suggesting that the vector could be used to deliver DNA sequences encoding tumor antigens for cancer immunotherapy.