This research sought to elucidate the molecular mechanism by which a recently described family of ion transport proteins, the cation diffusion facilitators (CDFs), transfer ions across biomembranes. Using the Escherichia coli homologs ZitB, and FieF, as well as CzcD from Cupriavidus metallidurans CH34 as models, the amino acids essential to CDF function were identified, and the transport behavior of ZitB and its homolog FieF, were described.Site-directed ZitB mutants were used to determine the necessity of individual amino acids. The mutation of several well-conserved acidic residues resulted in the loss of a ZitB-mediated zinc-resistant phenotype in the zinc-sensitive E. coli strain GG48. ZitB also complemented the potassium uptake deficient strain TK2420, suggesting that ZitB works as an antiporter, possibly allowing potassium into the cell while effluxing zinc.This result was further investigated in experiments using everted membrane vesicles(EMVs). Vesicles bearing ZitB accumulated 65Zn(II) in a NADH-dependent manner, with an apparent KM of 1 micromolar. This accumulation was inhibitable by the protonophore FCCP, suggesting CDF dependence upon the proton motive force. Similar results were obtained using both EMVs and proteoliposomes containing the CDF homologs CzcD, from C. metallidurans CH34, and FieF from E. coli. Despite facilitating 65Zn(II) uptakeinto EMVs, fieF transcription was zinc and iron-dependent, but independent of the ironuptake regulator Fur. FieF expression in trans complemented the iron-sensitive phenotypeof a strain lacking fur, causing it to accumulate less 55Fe than wild type. Reconstituted proteoliposomes containing FieF also accumulated less 55Fe than those without.This research confirms that CDF proteins likely depend on the cell's proton gradient, effluxing substrate in a metal:proton antiport arrangement. Substrate acquisition and throughput is facilitated by a set of acidic amino acids and histidines. The relatively lowapparent KM of ZitB suggests a homeostatic role for the protein; however, the iron-inducibilityof fieF hints at a role in iron detoxification, so the cellular functions of the CDF family may be quite diverse, even within the bacteria.