The membrane topology of the human Na(+)/H(+) exchanger isoform 1 (NHE1) was assessed by substituted cysteine accessibility analysis. Eighty-three cysteine residues were individually introduced into a functional cysteineless NHE1, and these mutants were expressed in the exchanger-deficient PS120 cells. The topological disposition of introduced cysteines was determined by labeling with a biotinylated maleimide in the presence or absence of preincubation with the membrane-impermeable sulfhydryl reagent, 2-trimethylammoniumethyl-methanethiosulfonate in streptolysin O-permeabilized or nonpermeabilized cells. We proposed a new model for the topology of NHE1 that is significantly different from the model derived from hydropathy analysis. In this model, NHE1 is composed of 12 transmembrane segments (TMs) with the N and C termini located in the cytosol. The large, last extracellular loop in the membrane domain of the original model was suggested to comprise an intracellular loop, a new transmembrane segment (TM11), and an extracellular loop in the new model. Interestingly, cysteines at 183 and 184 and at 324 and 325 mapped to intracellular loops connecting TMs 4 and 5 (IL2) and TMs 8 and 9 (IL4), respectively, were accessible to sulfhydryl reagents from the outside. Furthermore, exchange activities of two mutants, R180C and Q181C, within IL2 were markedly inhibited by external MTSET. These data suggest that part of IL2 or IL4 may be located in a pore-lining region that is accessible from either side of the membrane and involved in ion transport.