Target regions for posttranscriptional silencing of transgenes often reside in the 3′ region of the coding sequence, although there are exceptions. To resolve if the target region is determined by the gene undergoing silencing rather than by the structure of the transgene loci or the plant genetic background, we have performed detailed analyses of target regions in three spontaneously β-glucuronidase (GUS) silencing tobacco lines of different origin. From quantitative cosuppression experiments, we show that the main target region in all three tobacco lines is found within the 3′ half of the GUS coding region but upstream of the last 200 nt. The quantities of small (21–25 nt) RNAs homologous to 5′ or 3′ regions of the GUS coding sequence were found to correlate approximately with the target strength of the corresponding regions. These results suggest that transgene locus structure and plant genetic background are not major determinants of silencing target regions. We also show that virus-induced gene silencing (VIGS) of GUS in Nicotiana benthamiana is induced equally effectively with Potato virus X carrying either the 5′ or 3′ third of the GUS coding region. This indicates that both regions can act as efficient inducers as well as targets of posttranscriptional silencing, although the 3′ region is the predominant target region in the spontaneously silencing transgenic plant lines examined. Finally, we investigated spreading of the target region in the N. benthamiana plants undergoing VIGS. Surprisingly, only evidence for spreading of the target region in the 5′-3′ direction was obtained. This finding may help explain why the majority of target regions examined to date lie within the 3′ region of transgenes.