hCNT1, the first discovered of three human members of the SLC28 (CNT) protein family, is a Na+/nucleoside cotransporter with 650 amino acids. The potential functional roles of ten conserved aspartate and glutamate residues in hCNT1 were investigated by site-directed mutagenesis and heterologous expression in Xenopus oocytes. Initially, each of the ten residues was replaced by the corresponding neutral amino acid (asparagine or glutamine). Five of the resulting mutants showed unchanged Na+-dependent uridine transport activity (D172N, E338Q, E389Q, E413Q and D565N) and were not investigated further. Three were retained in intracellular membranes (D482N, E498Q and E532Q) and thus could not be assessed functionally. The remaining two (E308Q and E322Q) were present in normal quantities at cell surfaces, but exhibited low intrinsic transport activities. Charge replacement with the alternate acidic amino acid enabled correct processing of D482E and E498D, but not of E532D, to cell surfaces, and also yielded partially functional E308D and E322D. Relative to wild-type hCNT1, only D482E exhibited normal transport kinetics, while E308D/Q, E322D/Q and E498D displayed increased K50Na+ and/or Kmuridine values and diminished VmaxNa+ and Vmaxuridine values. E322Q additionally exhibited uridine-gated uncoupled Na+ transport. Together, these findings demonstrate roles for E308, E322 and E498 in Na+/nucleoside cotransport, and suggest locations within a common cation/nucleoside translocation pore. E322, the residue having the greatest influence on hCNT1 transport function, exhibited uridine-protected inhibition by p-chloromercuriphenyl sulfonate (PCMBS) and 2-aminoethyl methanethiosulfonate (MTSEA) when converted to cysteine.