Recent molecular cloning studies have identified four distinct members (NHE-1,-2,-3,-4) of the Na/H exchanger gene family that are expressed in a tissue-specific pattern. To examine some of their functional properties, full-length cDNAs for two of these isoforms, rat NHE-1 and NHE-3, were stably transfected in Na/H exchanger-deficient Chinese hamster ovary cells (AP-1) and assayed for transport activity by measuring amiloride-inhibitable H(+)-activated 22Na+ influx. Pharmacological analyses revealed that the activity of NHE-1 was substantially more sensitive to inhibition by amiloride and its analogues than NHE-3. Similarly, both isoforms were differentially sensitive to inhibition by cimetidine, clonidine, and harmaline; agents also known to inhibit the activity of the Na/H exchanger. Their rank order of potency for NHE-1 was cimetidine > harmaline > or = clonidine whereas the reverse order was observed for NHE-3. The isoforms were also distinguished by their kinetic properties. While the extracellular Na+ (Na+o) dependence of both isoforms showed simple, saturating Michaelis-Menten kinetics, NHE-1 exhibited a 2-fold lower affinity for Na+o than NHE-3, with apparent KNa values of 10.0 +/- 1.4 and 4.7 +/- 0.6 mM, respectively. In contrast to Na+o, intracellular H+ (H+i) activated both isoforms by a positive cooperative mechanism. However, NHE-1 had a 2-fold higher apparent affinity for H+i compared to NHE-3, with half-maximal activation values of pK 6.75 +/- 0.05 and 6.45 +/- 0.08, respectively. Other external cations also interacted with both exchangers. Li+o and H+o inhibited 22Na+ influx by both isoforms with similar kinetics. In contrast, K+o inhibited 22Na+ influx by NHE-1, but had no effect on NHE-3. Thus NHE-1 and -3 exhibited diverse functional properties when expressed in the same cell type. Furthermore, the functional properties associated with NHE-3 closely mimicked those described for the apical membrane Na/H exchanger of renal proximal tubules, suggesting that these proteins are molecularly identical.