Molecular cloning and expression have previously defined three members of the Na+/H+ exchanger (NHE) gene family. NHE1 and NHE2 are sensitive to inhibition by amiloride and its 5' amino alkyl-substituted analogues, whereas NHE3 is quite resistant to amiloride inhibition. Each of these exchangers has narrowly defined cation specificities for Na+ and Li+. Expression studies with NHE4 have not been as successful, with only a description of modest expression of activity (C. Bookstein, M. W. Musch, A. DePaoli, Y. Xie, M. Villereal, M. C. Rao, and E. B. Chang. J. Biol. Chem. 269: 29704-29709, 1994). We now report that NHE4 activity in stably transfected fibroblasts is activated by 4,4'-diisothiocyanostilbene-2,2'-disulfonic acid (DIDS), permitting functional characterization of this NHE isoform. The activating effect of DIDS was unique among the disulfonic stilbenes, and competition studies suggested a cross-linking mechanism. NHE4 is extremely resistant to amiloride and ethylisopropylamiloride inhibition and, unlike other NHE isoforms, affects K+/H+ exchange as well as Na+/H+ and Li+/H+ exchange. These findings demonstrate that NHE4 is a functionally distinct member of the NHE gene family and suggest a unique physiological role for this cation/H+ exchanger.