The authors describe a method for generating high-titer, helper-free retroviruses using transient transfection of the 293T cell line, which has been stably introduced with constructs expressing retroviral packaging functions. The resulting ectopic virus packaging cell line, BOSC 23, can produce infectious retroviruses at titers exceeding 10^6 infectious units/ml within 72 hours after CaPO4-mediated transfection. The system is free of helper virus and can produce high titers of retroviral vectors expressing genes that are difficult to propagate in stable producer lines. This method facilitates and extends the use of helper-free retroviral gene transfer and has potential applications in gene therapy. The BOSC 23 system is advantageous because it minimizes the detrimental effects of gene products on the producer cell line and does not produce detectable replication-competent virus.The authors describe a method for generating high-titer, helper-free retroviruses using transient transfection of the 293T cell line, which has been stably introduced with constructs expressing retroviral packaging functions. The resulting ectopic virus packaging cell line, BOSC 23, can produce infectious retroviruses at titers exceeding 10^6 infectious units/ml within 72 hours after CaPO4-mediated transfection. The system is free of helper virus and can produce high titers of retroviral vectors expressing genes that are difficult to propagate in stable producer lines. This method facilitates and extends the use of helper-free retroviral gene transfer and has potential applications in gene therapy. The BOSC 23 system is advantageous because it minimizes the detrimental effects of gene products on the producer cell line and does not produce detectable replication-competent virus.