To test the influence of a Nicotiana tomentosiformis repetitive sequence (R8.3) on transgene expression in N. sylvestris and in N. sylvestris-N. tomentosiformis hybrids, the R8.3 sequence was placed upstream of a nopaline synthase promoter (NOSpro)-NPTII reporter gene in a T-DNA construct. A number of transgenic N. sylvestris lines were produced and in most, the NPTII gene was expressed. In one line, however, the NPTII gene became silenced and methylated in the NOSpro region. The silenced locus was able to trans-inactivate and induce methylation of two stably expressed transgene loci comprising a similar construct. Nucleotide sequence analyses of the three transgene loci revealed that they each contained a single incomplete copy of the T-DNA, which had sustained deletions of varying sizes in the R8.3 region. Paradoxically, the R8.3 DNA upstream of the two active, unmethylated NOSpro-NPTII genes was highly methylated, whereas the R8.3 DNA upstream of the silenced, methylated NOSpro-NPTII gene was less methylated. The methylated portions of the R8.3 sequence corresponded to retroelement remnants. An active NOSpro-NPTII gene downstream of a nearly intact R8.3 sequence did not become methylated in N. sylvestris-N. tomentosiformis hybrids. Thus, methylation in the R8.3 sequence did not spread into adjoining transgene promoters and the effect of the R8.3 dispersed repeat family on transgene expression was negligible. The silencing phenomena observed with the three single-copy transgene loci are discussed in the context of other possible triggers of silencing.