Cell cultures latently infected with simian foamy virus type 3 (SFV-3) were established by suppressing lytic infection in Vero cells with 3'-azido-3'-deoxythymidine (AZT) and homologous antibodies (African green monkey serum immune to SFV-3). The resulting cell line, designated Vero-L, was shown to contain at least one copy per cell of SFV-3 DNA stably integrated at a defined site of the host cell genome. Sequencing of 669 bp at the integration site did not identify a coding region and revealed a 4-bp imperfect repeat in host cell DNA due to SFV-3 integration. Over 2 years of subcultivation, no spontaneous expression of proviral genes could be detected. However, the demethylating agent 5'-azacytidine reactivated lytic infection, proving conservation of the complete viral genome. Comparison of proviral DNA from latently and lytically infected cells supports the notion that methylation is instrumental in keeping SFV-3 infection in latency.