By transfecting fibroblast cells with an HIV-1-MN molecular clone with a deletion of the major packaging sequence, we have developed a stable HIV-1 packaging cell line, psi 422, psi 422 cells form syncytia with CD4-positive cells, correctly express HIV-1 structural proteins, and produce a large amount of mature particles with normal reverse transcriptase activity. Yet these particles, in which RNA was not detected by reverse transcriptase-PCR, are not infectious. When stably transfected with an HIV-1-based retroviral vector, the psi 422 cell line produces virions capable of transducing CD4-positive cells with high efficiency (up to 10(5) cells per ml). The availability of this stable noninfectious HIV-1 packaging cell line capable of generating high-titer HIV-1 vectors represents a new step toward the use of an HIV-1 delivery system in gene therapy.