The SLC30A5 gene encodes the bi-directional zinc transporter ZnT5, which is expressed at the apical enterocyte membrane (Cragg et al., 2005; Valentine et al., 2007). The presence of a CpG island in the SLC30A5 promoter suggests that expression may be regulated by DNA methylation. Aberrant DNA methylation has been implicated in the aetiology of cancer. Zinc dyshomeostasis in cancer cells, including abnormal zinc transporter expression (Taylor et al., 2007), has been reported to contribute to disease progression. We hypothesise that aberrant methylation of the SLC30A5 promoter in colon tumour cells may contribute to zinc dyshomeostasis through altered ZnT5 expression. A demethylated SLC30A5 promoter-reporter construct (Jackson et al., 2007) was generated by propagation through the methylase-deficient E. coli strain K12 ER2925 and an aliquot was methylated using SssI methylase. Caco-2 cells were transfected with these plasmids and β-galactosidase reporter gene activity was measured after 24 h. All data are stated as mean ± S.E.M in arbitrary units. The reporter gene was expressed from the unmethylated, but not the methylated, construct (P<0.001 by one way ANOVA followed by Tukey’s multiple comparison test; 1.00 ± 0.03 for the unmethylated construct, 0.29 ± 0.05 for the methylated construct, 0.18 ± 0.03 for negative control (vector only); n = 23), indicating that methylation of the SLC30A5 promoter suppresses gene expression. In contrast, reporter gene expression from an equivalent construct incorporating a promoter without a CpG island (mouse Srpx2) was not affected by methylation (P>0.05 by Student’s t-test; 1.00 ± 0.18 for the unmethylated construct, 0.87 ± 0.05 for the methylated construct; n = 6). Methylation of the SLC30A5 promoter in 23 samples of human colon tumour tissue and from paired samples taken at least 10 cm away from the tumour site was measured by Pyrosequencing. Methylation was consistently lower in tumour than in corresponding normal tissue and this effect reached statistical significance in 3 of the 21 CpG sites analysed (P<0.05 by Student’s paired t-test; 69% compared with 51% (mean) for the CpG site at position -753 relative to the start of transcription, 91% compared with 62% for the site at position -606, and 75% compared with 58% for the site at position -458, n=5-23). We have established in vitro that methylation of the human SLC30A5 promoter region results in reduced expression of an associated reporter gene and that methylation of CpG sites in the SLC30A5 promoter in colon tumour tissue was consistently lower than in adjacent normal tissue. A proposed, but unproven, resulting increase in ZnT5 expression may contribute to zinc dyshomeostasis in tumour cells and may be a factor contributing to cancer development and/or progression. Funded by BBSRC grants BB/F019637/1 and BB/E007457/1