The ZIP (ZRT-, IRT-like Protein) protein ZupT from Escherichia coli is a transporter with a broad substrate range. Phenotypic and transport analysis showed that ZupT, in addition to Zn(II), Fe(II) and Co(II) uptake, is also involved in transport of Mn(II) and Cd(II). Competition experiments with other substrate cations suggested that ZupT has a slight preference for Zn(II) and kinetic parameters for Zn(II) in comparison to Co(II) and Mn(II) transport support this observation. Metal uptake into cells by ZupT was optimum at near neutral pH and inhibited by ionophores. Bicarbonate or other ions did not influence metal-uptake via ZupT. Amino acid residues of ZupT contributing to substrate specificity were identified by site directed mutagenesis. ZupT with a H89A exchange lost Co(II) and Fe(II) transport activity, while the S117V mutant no longer transported Mn(II). ZupT with E152D was impaired in overall metal uptake but completely lost its ability to transport the substrates Zn(II) and Mn(II). These experimental findings expand our knowledge on the substrate specificity of ZupT and provide further insight into the function of ZupT as a bacterial member of the vastly distributed and important ZIP family.