Copper is an essential metal, involved in a number of key enzymes in human metabolism. Dietary copper import takes place in the duodenum and requires that the metal is present in its reduced cuprous form. To date the mechanisms involved in the reduction of Cu (II) to Cu (I) are unclear. Like copper, iron must also be present in the reduced Fe (II) form before it becomes bioavailable. This is achieved in part by the presence of the duodenal ferric reductase Dcytb [1]. Given the overlap between copper and iron metabolism [2], the aim of this study was to determine whether Dcytb might also act as a cupric reductase. Dcytb was isolated from mouse duodenal mRNA, PCR amplified, and ligated into a pEGFP-N1 vector (Clontech, Palo Alto, USA) to permit to addition of a carboxy terminal enhanced green fluorescent protein (EGFP) tag. The Dcytb-EGFP product was subsequently excised and inserted into pTRE2hyg vector (Clontech) which contains a tetracycline responsive element upstream of a minimal CMV promoter to control expression of Dcytb-EGFP. The plasmid was transfected into MDCK Tet-off cells (Clontech) which allows Dcytb-EGFP expression to be switched off in the presence of doxycyclin (10ng/ml culture medium for 4 days). Stably transfected cells were used to study cupric reductase activity using bathocuprionedisulfonate (BCS). The formation of the Cu (I)-BCS complex was monitored by the change in absorbance at 482nm. Standard curves were generated to convert the absorbance value into pmoles of copper reduced. All reactions were performed in the dark at 37C. Statistical analysis was performed using one-way ANOVA and Tukey’s post-hoc test and differences were considered significant at P<0.05. Data are mean ± SEM. MDCK cells over-expressing Dcytb-EGFP protein reduced significantly greater amounts of copper (transfected, 0.70 ± 0.02 pmol/μg cell protein/min; untransfected, 0.07 ± 0.01 pmol/μg cell protein/min; P<0.001, n = 6 in each group). Reductase activity was significantly diminished in the presence of doxycyclin (0.37 ± 0.01 pmol/μg cell protein/min; P<0.01, n = 6). These data provide the first evidence that, in addition to its well-characterised role as a ferric reductase, Dcytb also function as a cupric reductase in vitro. We are exploring further the role of this enzyme in copper metabolism.