The effect of a transmembrane pH gradient on the ouabain, bumetanide, and phloretin resistant H + efflux was studied in rabbit erythrocytes. Proton equilibration was reduced by the use of DIDS (125 pM) and acetazolamide (1 mM). H + efflux from acid loaded erythrocytes (pH, = 6.1) was measured in a K + (145 mM) medium, pHo = 8.0, in the presence and absence of 60 pM 5,N,Ndimethyl-amiloride (DMA). The H + efflux rate in a K+-containing medium was 116.38 4.5 mmol/l cell x hr. Substitution of Na; for K; strongly stimulated H + efflux to 177.89 & 7.9 mmol/l cell x hr. The transtimulation of H + efflux by Na; was completely abolished by DMA falling to values not different from controls with an ID,o of about 8.6 x IO-'M. The sequence of substrate selectivities for the external transport site were Na > > > Li > choline, Cs, K, and Glucamine. The transport system has no specific anion requirement, but is inhibited by NO;. The DMA sensitive H + efflux was a saturable function of "a+],, with an apparent Km and V, , , of about 14.75 1.99 mM and 85.37 f 7.68 mmolll cell x hr, respectively. However, the Nazdependent and DMA-sensitive H+ efflux was sigmoidally activated by [H+Ii, suggesting that H: interacts at both transport and modifier sites. An outwardly directed H+ gradient (pHi 6.1, pH = 8.0) also promoted DMA sensitive Na+ entry (61.2 f 3.0 mmolll cell x hr) which was abolished when pH, was reduced to 6.0. The data is therefore consistent with the presence of a Na+/ H + exchange system in rabbit erythrocytes.