Transgene silencing in plants is most often dependent on homologous sequences, e.g. tandemly repeated T-DNAs. We have identified an Arabidopsis line (ex2–4 line 4) displaying silencing of the T-DNA-born nptII gene. This line contains a truncated copy of the T-DNA encompassing the nptII gene with its nos promoter adjacent to an intact T-DNA copy. The orientation of the intact and the truncated copies preclude the generation of a double-stranded nptII transcript. Therefore, we have investigated the genomic landscape surrounding T-DNA insertion in the silenced ex2–4 line 4 and five single-copy ex2–4 lines without silencing in search of features that might explain the silencing phenomenon. GC content, putative matrix-attachment regions and transcriptional interference from neighbouring genes could all be ruled out as major causes of silencing. Bisulphite sequencing revealed de novo methylation of the nos promoter both in non-silenced and silenced plants of this line, thus silencing was not correlated to DNA methylation level. Also, the methylation pattern deviated from that characteristic for RNA-mediated DNA methylation and silencing. Our data therefore suggest that ex2–4 line 4 represents a case where silencing is due to DNA-DNA pairing, i.e. pairing between the intact T-DNA and the adjacent truncated, inverted T-DNA copy.