The topology of the plasma membrane Na(+)/Ca(2+) exchanger of cardiac muscle, NCX1, is uncertain. Biochemical analyses have indicated the presence of 9 transmembrane segments (TMSs) whereas the recent crystal structure of a prokaryotic homologue has 10 TMSs. The discrepancy is towards the C-terminus of the proteins where the prokaryotic homologue has an additional TMS8. To resolve this apparent disagreement, we re-assessed the topology of the C-terminal TMSs of NCX1. We examined the ability of internal or external cysteine residues in the N-terminal portion of NCX1 to crosslink with cysteine residues, of uncertain orientation, in the C-terminal portion of the protein. The results strongly support a model of NCX1 with 10 TMSs as found in the prokaryotic homologue.