- Background The Na + /Ca 2 + /Li + exchanger (NCLX) is a member of the Na + /Ca 2 + exchanger family. NCLX is unique in its capacity to transport both Na + and Li + , unlike other members, which are Na + selective. The major aim of this study was twofold, i.e., to identify NCLX residues that confer Li + or Na + selective Ca 2 + transport and map their putative location on NCLX cation transport site. Method We combined molecular modeling to map transport site of NCLX with euryarchaeal H + /Ca 2 + exchanger, CAX_Af, and fluorescence analysis to monitor Li + versus Na + dependent mitochondrial Ca 2 + efflux of transport site mutants of NCLX in permeabilized cells. Result Mutation of Asn149, Pro152, Asp153, Gly176, Asn467, Ser468, Gly494 and Asn498 partially or strongly abolished mitochondrial Ca 2 + exchange activity in intact cells. In permeabilized cells, N149A, P152A, D153A, N467Q, S468T and G494S demonstrated normal Li + /Ca 2 + exchange activity but a reduced Na + /Ca 2 + exchange activity. On the other hand, D471A showed dramatically reduced Li + /Ca 2 + exchange, but Na + /Ca 2 + exchange activity was unaffected. Finally, simultaneous mutation of four putative Ca 2 + binding residues was required to completely abolish both Na + /Ca 2 + and Li + /Ca 2 + exchange activities. Conclusions We identified distinct Na + and Li + selective residues in the NCLX transport site. We propose that functional segregation in Li + and Na + sites reflects the functional properties of NCLX required for Ca 2 + exchange under the unique membrane potential and ion gradient across the inner mitochondrial membrane. General significance The results of this study provide functional insights into the unique Li + and Na + selectivity of the mitochondrial exchanger. This article is part of a Special Issue entitled: ECS Meeting edited by Claus Heizmann, Joachim Krebs and Jacques Haiech.