Derivatives of the cauliflower mosaic virus 35S promoter lacking CG and CNG methylation targets were constructed and used to direct transcription of reporter gene constructs in transiently transformed protoplasts. Such methylation-target-free (MTF) promoters, although weaker than the 35S promoter, retain significant activity despite mutation of the as-1 element. The effect of methylation on gene expression in MTF- and 35S-promoter driven constructs was examined. Even when the promoter region was free of methylation targets, reporter gene expression was markedly reduced when cytosine residues in CG dinucleotides were methylated in vitro prior to transformation. Mosaic methylation experiments, in which only specific parts of the plasmids were methylated, revealed that methylation of the coding region alone has a negative effect on reporter gene expression. Methylation nearer the 5' end of the coding region was more inhibitory, consistent with inhibition of transcription elongation.