Hartnup disorder is an autosomal recessive disorder caused by mutations in the B°AT1 (Slc6a19) gene encoding the major transport system for neutral amino acids in the intestinal brush-border membrane. B°AT1 protein is expressed at the apical membrane of enterocytes in increasing amounts along the crypt-villus axis. Previously, we showed that HNF1a and HNF4a bind to the Slc6a19 promoter up-regulating transcription. Sox9, by contrast, suppressed the promoter activity induced by HNF1a and HNF4a. Sox9 is a transcription factor known to be involved in the differentiation of stem cells into mature enterocytes, and is highly expressed in the crypt region, but absent at the villus tip. Thus Sox9 expression could explain the gradient of B°AT1 in the intestine. However, despite HNF1a and HNF4a expression in the liver, B°AT1 is not expressed, while Sox9 is absent. As a result we considered DNA methylation as a mechanism to regulate B°AT1 expression. DNA methylation of a transcription factor binding sites can prevent transcription factor from binding to the DNA. Kikuchi et al (2010) suggested that HNF1a binding to the B°AT1 promoter could be prevented by methylation in the liver. However, they observed particular methylation in the liver at position -1080, which is 940 bp upstream of the HNF1a binding site. In this study we investigated the DNA methylation status of the B°AT1 promoter, using bisulfate sequencing. DNA methylation was determined in a 1.2 kb region upstream of the B°AT1 transcriptional start site in liver, kidney and crypt-villus preparations. From each experiment a minimum 10 independent clones were sequenced. Twenty CpG sites were analysed and found to be differentially methylated in the chosen tissues. We found that CpG dinucleotides around HNF1a, HNF4a and TATA binding sites were hypomethylated in the villus, whilst being hypermethylated in the crypt and in liver tissue. These results indicate that DNA methylation might play a role together with Sox9 in the repression of B°AT1 expression in the crypt, whereas in the liver cells DNA methylation itself might be sufficient.