Neutral endopeptidase 24.11 (NEP), a cell-surface enzyme expressed by epithelial cells that cleaves and inactivates biologically active small peptides, is downregulated in various cancers. NEP is encoded by a gene that contains a CpG island in the promoter region, whose hypermethylation appears related to decreased expression. Altered expression of NEP has also been reported in human hepatocellular carcinoma (HCC), suggesting its possible role in hepatocarcinogenesis. To elucidate the status of NEP in HCC, methylation in the promoter region of the gene that encodes NEP in male Fischer 344 rats with HCC, induced by a choline-deficient, l-amino acid-defined diet, was investigated by methylation-specific polymerase chain reaction, combined bisulfite restriction analysis, and bisulfite genomic sequencing. These analyses together showed the promoter to be frequently methylated in HCC in contrast to its unmethylated status in normal liver, the degree of methylation being inversely related to the level of mRNA expression evaluated by reverse transcription-polymerase chain reaction (P = 0.031). In two rat liver cell lines, RLC-16 and RLC-27, the promoter was heavily methylated and NEP mRNA expression was negative. However, administration of 5-aza-2'-deoxycytidine caused NEP expression, suggesting that methylation of CpG is a factor regulating transcriptional expression. Together with the data from microarray analyses performed previously using the same animal model, the current results suggest that reduced expression of NEP or other ectopeptidases could impact on molecules involved in signal-transducing systems, including G-protein coupled receptors, via modified turnover of extracellularly active small peptides.