The process of growing and transducing large quantities of human primary peripheral blood lymphocytes (PBLs) with high gene transfer efficiency continues to be one of the major challenges for clinical and experimental gene therapy. Toward developing a clinical trial of lymphocyte gene therapy for mucopolysaccharidosis type II (i.e., Hunter syndrome), we investigated a novel method that exploited the innate capability of a hollow-fiber bioreactor system to filter large quantities of vector supernatant and facilitate transduction. An aliquot (5 x 107) of PBL apheresis product was precultured in a gas-permeable culture bag or a bioreactor, and then transduced with a retroviral vector L2SN containing the iduronate-2-sulfatase (IDS) and neomycin resistance genes. We observed that the total number of PBLs could be expanded up to 187-fold, yielding up to 1010 cells at the end of a 7-day culture period. The multiplicity of infection could be increased (up to 20-fold) by ultrafiltrating a large volume of vector supernatant through the semipermeable membrane of this system. A high level of transduction efficiency (up to 57%) was achieved, resulting in IDS enzyme activity as high as 1250 U/mg/hr in transduced PBL(MPS) 15 days after transduction. This level was markedly increased from that of nontransduced cells (<3 U/mg/hr) and was even greater than that of normal PBLs (mean, 809; n = 10). After 12 days of G418 selection, PBL(MPS) transductants exhibited a proviral IDS enzyme level approximately threefold higher than that in normal PBLs. These results indicated that the hollow-fiber bioreactor could be used to culture and transduce human primary PBLs in clinically useful quantities with relatively high gene transfer efficiency and transgene expression.