L1 (LINE-1) is an interspersed non-LTR retrotransposon and several genetic defects caused by L1 transposition have been reported. L1 is thus considered as a potential mutagen. However, this potentially hazardous insertional event seems to be rare in spite of the presence of 3000 or more L1 elements of full or nearly full length in the human genome. Thus there must exist a mechanism(s) for repressing the expression of most, if not all, L1 elements. Some studies suggested that methylation plays a major role in the repression of L1 expression. However, no direct evidence has been presented and further study is required to draw a conclusion. We thus studied the effect of methylation on L1 transcription in vivo and in vitro. Transfection of plasmid which contained a L1 promoter linked to cat gene into HeLa cells showed that methylation did repress the L1 promoter activity. In vitro transcription studies using mutagenized templates indicated that methylation of the first seven CpGs in L1 promoter, particularly four CpGs at +52, +58, +61 and +70 was essential for the inhibition. These results suggest that there exists a mechanism to regulate the L1 transcription through the region-specific methylation.