Kidneys play a key role in zinc balance. The portion of Zn(II) that enters the glomerular filtrate is efficiently reabsorbed along the nephron through a mechanism yet to be identified. We used the Zn(II)-specific fluorophore Zinquin to visualize intracellular Zn(II) accumulated in the kidney epithelium and compared it with the intracellular localization of the vesicular zinc transporter ZnT4 both in vivo and in vitro. The Madin-Darby canine kidney (MDCK) cell line, stably overexpressing rat ZnT4, was used as a tissue culture model of the kidney epithelium. Zinquin labeling of MDCK cells revealed rapid internalization of Zn(II) and compartmentalization in intracellular bodies interspersed throughout the cytoplasm. In polarized kidney cells, ZnT4 protein was localized on the membrane of intracellular vesicles concentrated around the nucleus, mostly on the basal side. Results of double stainings demonstrated that ZnT4-containing vesicles do not overlap with Zn(II) bodies. Zinquin fluorescence, confirmed by autometallography in rat kidney, indicated that consistent with its physiological role, the central glomerulus was weakly stained, whereas the epithelium that lines convoluted tubules was strongly labeled. Double staining of rat kidney with Zinquin and anti-ZnT4 antibodies confirmed the in vitro observations, as Zinquin fluorescence appeared to be distinct from ZnT4 immunofluorescence. To gain further insight into which of the known zinc transporters might be involved in Zn(II) metabolism in the kidney, we have also characterized by RT-PCR the expression of other proteins involved in Zn(II) transport. All of the mRNAs examined [ZnT1, -T2, -T4, and human Zrt, Irt-like protein 1 (hZIP1)], with the exception of hZIP2, were present in adult rat kidney.