We have previously reported that the long terminal repeat (LTR) region of feline leukemia viruses (FeLVs) can enhance expression of certain cellular genes such as the collagenase IV gene and MCP-1 in trans (S. K. Ghosh and D. V. Faller, J. Virol. 73:4931-4940, 1999). Genomic DNA of all healthy feline species also contains LTR-like sequences that are related to exogenous FeLV LTRs. In this study, we evaluated the cellular gene transactivational potential of these endogenous FeLV LTR sequences. Unlike their exogenous FeLV counterparts, neither nearly full-length endogenous FeLV molecular clones (CFE-6 and CFE-16) nor their isolated LTRs were able to activate collagenase IV gene or MCP-1 expression in transient transfection assays. We had also demonstrated previously that production of an RNA transcript from exogenous FeLV LTRs correlates with their transactivational activity. In the present study, we demonstrate that the endogenous FeLV LTRs do not generate LTR-specific RNA transcripts in the feline embryo fibroblast cell line AH927. Furthermore, infection of AH927 cells by an exogenous FeLV subgroup A virus did not induce production of such LTR-specific transcripts from the endogenous proviral genomes, although the LTR-specific transcripts from the exogenous virus were readily detected. Finally, LTR-specific transcripts were not generated in BALB/3T3 cells transiently transfected with isolated CFE-6 LTR, in contrast to transfections with LTRs from exogenous viruses. Our data thus suggest that the inability of endogenous FeLV LTRs in gene transactivation is not due to cell line specificity or presence of any upstream inhibitory cis-acting element. Endogenous, nonleukemogenic FeLV LTRs, therefore, do not transactivate cellular gene expression, and this property appears to be specific to exogenous, leukemogenic FeLVs.