Summary: Transgenic tobacco plants carrying post‐transcriptionally, silenced (L) or conditionally high‐expressing (Hc) cauliflower mosaic virus 35S promoter:β‐glucuronidase (GUS) transgenes were tested for their ability to support replication of a potato virus X derivative with an inserted GUS coding sequence (PVX.GUS). PVX.GUS accumulated to high levels on non‐transformed tobacco. In contrast, both L and Hc tobacco were resistant to PVX.GUS. Another line, 271, carries a 35S‐driven transgene that can down‐regulate transcription from any other 35S promoter in trans. When the 271 locus was introduced into the L and Hc lines, virus resistance was suppressed. The L tobacco line also trans‐inactivated transient GUS expression from an extra‐chromosomal T‐DNA delivered by Agrobacterium. This trans‐inactivation was not observed when the 271 locus was present. In addition, it was found that the 271 locus suppressed the virus resistance normally conferred by a 35S‐driven transgene derived from the PVX replicase open reading frame. These results indicate that sense transcription is required for a transgene to condition homology‐dependent virus resistance and post‐transcriptional trans‐inactivation.