Lentivectors have drawn considerable attention recently and become important delivery vehicles for gene transfer manipulation. By Transiently co-transfecting 293T packaging cells with three DNA plasmids system encoding lentivector constituents, a protocol for bulky preparation of human immunodeficiency virus type-1 ( HIV-1)-based defective lentivector with high titer has been established. Transient co-transfection of 293T packaging cells resulted in production of high-titer vector (1.1 x 10 IU/ml), which can be further concentrated over 100-fold through a single step centrifugation. The vector was capable of efficiently transducing a variety of cells from both primate and non-primate sources, including of human T-lymphoblastoid cell line. Long-term culture of vector transduced cells showed a stable expression of foreign gene over 18 months detected by RT-PCR. Assessment of potential generation of replication-competent virus revealed no detection of p24 antigen protein or infectious particles in vector-transduced cells.