A primary biosafety issue of retroviral vector-mediated gene therapy is the genetic instability of retroviral vectors. Reverse transcription of vector RNA genome is initiated by viral reverse transcriptase (RT) in a virion particle after infection of a target cell. During reverse transcription, abnormal template switches between vector and occasionally co-packaged helper virus in a virion particle can therefore enable helper virus to regain replication elements from the vector and revert to replication-competent retrovirus (RCR). This research was undertaken to study the origins of RT enzyme activities and test the hypothesis that RT enzyme activities are contributed by both exogenous RT, which is imported by the re-infection of vector virions, and endogenous RT, which is provided by intracellular vector virion particles. Superinfection of vector in vector producer cells (VPC) can increase the number of integrated vectors in VPC, and is mainly caused by decreased Env-receptor interference, a consequence of helper virus gene inactivation by host DNA methylation at the 5' LTR promoter region. Suppression of helper virus gene expression by DNA methylation also reduces vector production. A chimeric retroviral helper virus combined with picornavirus IRES (internal ribosome entry site) sequence and a selection marker was therefore constructed to eliminate DNA methylated helper virus from cell populations to maintain active gene expression and enhance Env-receptor interference. Packaging cells established by this chimeric helper virus exhibit high titer production without detectable superinfection. In addition to exogenous RT activities imported by superinfection, significant endogenous RT activities (0.2% to 7.8% of exogenous RT activity), which originates from viral precursor protein Pr180gag-pol, were also demonstrated by intracellular retrotransposition of a retroviral vector in VPC. Retrotransposed vectors can integrate into different chromosomal locations to increase the plasticity of the VPC genome as observed from superinfected vectors. The outcomes from this study provide important insights for further strategies designed to prevent RCR formation by inactivation of RT activity. Demonstration of intracellular retrotransposition of retroviral vectors in VPC may reveal an alternative replication pathway of retrovirus.