Vectors derived from murine retroviruses have been used extensively for gene transfer in both preclinical and clinical studies. Retroviruses are small RNA viruses that replicate through a double-stranded DNA intermediate. The ability of retroviral vectors to integrate efficiently into the host DNA of infected cells, resulting in stable gene expression, makes them well suited for certain gene therapy applications. The majority of the applications using retroviral vectors have involved ex vivo methods in which cells are genetically modified in culture prior to introduction into the animal (1 ,2 ). However, the recent improvement in methods for production and concentration of retroviruses now allows for direct, in vivo applications (see Notes 1 and 2 ). This chapter will give a brief background on Moloney Murine Leukemia Virus- (MLV) based retroviruses and describe how to construct, produce, and titer replication-defective retroviral vectors.