The application of antisense transgenes in plants is a powerful tool to inhibit gene expression. The underlying mechanism of this inhibition is still poorly understood. High levels of antisense RNA (as-RNA) are expected to result in strong silencing but often there is no clear correlation between as-RNA levels and the degree of silencing. To obtain insight into these puzzling observations, we have analyzed several petunia transformants of which the pigmentation gene chalcone synthase (Chs) is post-transcriptionally silenced in corollas by antisense (as) Chs transgenes. The transformants were examined with respect to the steady-state as-RNA level, transcription level of the as-transgenes, the repetitiveness and structure of the integrated T-DNAs, and the methylation status of the transgenes. This revealed that the transformants can be divided in two classes: the first class contains a single copy (S) T-DNA of which the as-Chs gene is transcribed, although several-fold lower than the endogenous Chs genes. As there are not sufficient as-RNAs to degrade every mRNA, we speculate that silencing is induced by double-stranded RNA. The second class contains two T-DNAs which are arranged as inverted repeats (IRs). These IR loci are severely methylated and the as-Chs transgenes transcriptionally barely active. The strongest silencing was observed with IR loci in which the as-Chs transgenes were proximal to the centre of the IR. Similar features have been described for co-suppression by IRs composed of sense Chs transgenes, suggesting that silencing by antisense IRs also occurs by co-suppression, either via ectopic DNA pairing or via dsRNA.