Two transgenes inserted into different genomic positions can co-inactivate each other when they share homologous sequences while each of the two homologous transgenes is stably expressed in the absence of a second homologous copy. To evaluate the efficiency of such homology-dependent gene silencing (HDGS) effects, we have produced 19 tobacco transformants that contained a stably expressed NPTII transgene inserted into a single genomic locus, and have analysed the stability of each transgene in the presence of a second stably expressed homologous transgene. All transformants shared the coding region of the NPTII gene but individual transformants differed in transgene copy number, expression levels and in the continuity of the transgene homology due to the insertion of introns into the NPTII region as well as the use of different promoters and terminators for the design of the transgene constructs. We generated 189 progeny populations representing all possible dual combinations among the 19 lines and analysed the kanamycin resistance of 400 seedlings of each cross. Our data show (1) that gene silencing occurs at a relative low frequency when transgenic loci sharing an homology at the coding sequence level are combined, and (2) that neither the variation of this homology by insertion of introns in the coding sequence, or by changing the promoter and terminator of the construct, nor the variation in the expression level of the transgene, are decisive parameters modifying the efficiency of co-silencing between two NPTII transgenes.