The gut and liver share a common embryological origin. The gene encoding the gut hormone neurotensin/neuromedin N (NT/N) is expressed in the adult small bowel, and NT/N is transiently expressed in the fetal liver, suppressed in the adult liver, and reexpressed in certain liver cancers. In our present study, we found that the NT/N gene was expressed at high levels in the human hepatoma cell line Hep 3B but was not expressed in Hep G2 cells. To further determine the mechanisms regulating NT/N expression, we performed Southern blotting and gene cloning techniques. Neither alteration nor mutation of the NT/N gene was responsible for this differential NT/N expression pattern. Human NT/N promoter constructs were transfected into either Hep 3B or Hep G2. Both cell lines supported NT/N transcription, indicating that the absence of NT/N expression in Hep G2 cells was due to mechanisms other than the absence of positive transcription factors. The role of DNA methylation was next assessed. Methylation of NT/N promoter constructs in vitro resulted in a 67-fold reduction in promoter activity, whereas treatment with the demethylating agent 5-azacytidine induced NT/N expression in Hep G2 cells, thus suggesting that DNA methylation plays a role in the expression of the gut endocrine gene NT/N. Defining the mechanisms regulating NT/N expression in these hepatic-derived cell lines will provide not only a better understanding of cell-specific and developmental regulation of a gut endocrine gene but also possible insight into liver cell lineage patterns and the derivation of certain hepatocellular cancers.