FTO-2B rat hepatoma cells acquired mouse VL30 retrotransposon(s) when infected with Moloney murine leukemia virus (MoMLV) recombinant retroviruses produced from psi 2 cells. The VL30 provirus was integrated into the rat genome, expressed at high levels, and its transcription induced 40-fold by dexamethasone, VL30 RNA was detected in hepatoma cells even without selection for the expression of the amino-3'-glycosyl phosphotransferase (neo) gene, which was co-transferred with a MoMLV retrovirus. However, the extent of transfer of the VL30 RNA was inversely related to the titer of the MoMLV recombinant retrovirus. The restriction map analysis of the transferred VL30 provirus was identical to the mouse VL30s of the NVL subfamily which is known to be a significant fraction of the transcriptionally active VL30 subset. Additionally, the regenerating liver from an adult rat, which was infected with a defective MoMLV-derived retrovirus, expressed VL30 RNA. These results indicate that great care should be given to the transfer of unwanted passengers, like VL30, present in retroviral packaging cell lines like the psi 2 cells, which are currently being used for gene therapy.