Pronounced variability of transgene expression and transgene silencing are commonly observed among independent plant lines transformed with the same construct. Single-copy T-DNA lines harboring reporter genes of various kind and number under the control of a strong promoter were established in Arabidopsis thaliana for a comprehensive analysis of transgene expression. Characterization of 132 independent transgenic lines revealed no case of silencing as a result of site of T-DNA integration. Below a certain number of identical transgenes in the genome, gene copy number and expression were positively correlated. Expression was high, stable over all generations analyzed, and of a comparable level among independent lines harboring the same copy number of a particular transgene. Conversely, RNA silencing was triggered if the transcript level of a transgene surpassed a gene-specific threshold. Transcript level-mediated silencing effectively accounts for the pronounced transgene expression variability seen among transformants. It is proposed that the RNA sensing mechanism described is a genome surveillance system that eliminates RNA corresponding to excessively transcribed genes, including transgenes, and so plays an important role in genome defense.