The Tensins are a family of intracellular cytoskeleton-interacting proteins that are involved in the regulation of cell motility and migration. Downregulation of Tensins has been observed in several cancers, indicating that it may be advantageous for tumour progression. In this study, an epigenetic basis for the observed downregulation of Tensin3 in human renal cell carcinoma (RCC) has been investigated, specifically the methylation state of the TNS3 gene promoter. The aims of this study were to: 1. Identify and validate a functional promoter for the human TNS3 gene that contains a CpG island within it; 2. quantify the methylation level of this region in RCC; 3. determine whether expression of Tensin3 could be altered through DNA demethylation treatment. Bioinformatic analysis revealed there to be a putative promoter for the human TNS3 gene, housing an 826-bp CpG island. A luciferase reporter assay demonstrated a functional minimal promoter activity for a 2000 bp sequence containing this island. For quantification of methylation in the TNS3 promoter, genomic DNA from RCC patients (tumour and adjacent non-tumour) and a normal control group were analysed by bisulphite conversion followed by pyrosequencing analysis. This enabled quantitative determination of DNA methylation of individual CpG dinucleotides within the TNS3 gene promoter, out of a total of 43 analysed. Across the entire CpG stretch, RCC DNA showed significantly higher methylation level than non-tumour kidney DNA and normal control DNA (tumour n=12, non-tumour n=3; normal n=12; P<0.01, tumour vs. non-tumour; P<0.0001, tumour vs. normal). Out of the CpGs analysed, two CpG dinucleotides, CpGs 2 and 8, showed the most pronounced increases in methylation in tumour samples (P<0.0001). Furthermore, CpG 2 methylation negatively correlated with Tensin3 gene expression levels in RCC samples (P<0.005). In addition, pharmacological demethylation of cultured HK2 kidney cells with 5-aza-2’deoxycytidine caused a threefold upregulation of Tensin3 expression as measured by qRT-PCR. In conclusion, these results reveal a differential methylation pattern in the TNS3 promoter region occurring in human RCC, suggesting at least in part an epigenetic basis for the observed aberrant downregulation of Tensin3 in this disease.