Methylation has often been correlated with transcriptional inhibition of genes transcribed by polymerase II, but its role on polymerase III genes is less well understood. Using the genomic sequencing technique, we have analysed the methylation pattern of the different 5S-rDNA arrays of the Arabidopsis genome. Every cytosine position within the 5S sequence is highly methylated whatever the context – CpG, CpNpG or non-symmetrical. The methylation pattern of both transcribed and non-transcribed 5S units is similar, with no preferential methylated or unmethylated site. These results, taken together with 5-azacytidine treatments and in vitro transcription experiments using methylated 5S templates, demonstrate that 5S rRNA gene transcription is not inhibited by methylation. Non-transcribed 5S arrays are more subject to transition mutations resulting from deamination of 5-methylcytosines, leading to CpG depletions and an increasing A + T content. As there were no detectable differences in methylation, this implies more efficient repair and/or selection pressure in transcribed 5S-blocks.