Retroviral vector producer cells (VPC) have been considered genetically stable. A clonal cell population exhibiting a uniform vector integration pattern is used for sustained vector production. Here, we observed that the vector copy number is increased and varied in a population of established LTKOSN.2 VPC. Among five subclones of LTKOSN.2 VPC, the vector copy number ranged from 1 to approximately 29 copies per cell. A vector superinfection experiment and Northern blot analysis demonstrated that suppression of helper virus gene expression decreased Env-receptor interference and allowed increased superinfection. The titer production was tightly associated with helper virus gene expression and varied between 0 and 2.2 × 10⁵ CFU/ml in these subclones. In one analyzed subclone, the number of integrated vectors increased from one copy per cell to nine copies per cell during a 31-day period. Vector titer was reduced from 1.5 × 10⁵ CFU to an undetectable level. To understand the mechanism involved, helper virus and vectors were examined for DNA methylation status by methylation-sensitive restriction enzyme digestion. We demonstrated that DNA methylation of helper virus 5' long terminal repeat occurred in approximately 2% of the VPC population per day and correlated closely with inactivation of helper virus gene expression. In contrast, retroviral vectors did not exhibit significant methylation and maintained consistent transcription activity. Treatment with 5-azacytidine, a methylation inhibitor, partially reversed the helper virus DNA methylation and restored a portion of vector production. The preference for methylation of helper virus sequences over vector sequences may have important implications for host-virus interaction. Designing a helper virus to overcome cellular DNA methylation may therefore improve vector production. The maintenance of increased viral envelope-receptor interference might also prevent replication-competent retrovirus formation.