Although the three mammalian Na+-Ca2+ exchangers share considerable amino acid sequence homology, they exhibit substantial immunosuppressive drug specificity. We have shown that cyclosporin A (CsA) treatment of NCX1-, NCX2-, or NCX3-transfected HEK 293 cells and non-transfected H9c2, L6, and aortic smooth muscle cells, which express NCX1 protein naturally, reduces NCX surface expression and transport activity but has no impact on total cell NCX protein. Similar effect on functional expression of NCX1 protein can be obtained also without CsA treatment by knockdown of cell cyclophilin A (CypA), one of the cellular receptor of CsA. This suggests that CypA has a role in acquisition of function competence of NCX1 protein. Unlike CsA treatment, which affects the functional expression of all three mammalian NCX proteins similarly, FK506 and rapamycin treatment modulates only the functional expression of NCX2 and NCX3 proteins. FK506 reduces NCX2 and NCX3 surface expression and transport activity without affecting cell NCX protein. Rapamycin reduces NCX2 and NCX3 transport activity but has no effect on their surface expression or total cell NCX protein expression suggesting that, although it shares a common receptor FKBP with FK506, its mode of action follows a different pathway. We are showing now that the large cytosolic loop of NCX1, NCX2, and NCX3 is involved in acquisition of immunosuppressive drug specificity: truncation of the large cytosolic loop of NCX1 renders the protein sensitive to FK506. Exchange of the large cytosolic loop of NCX3 with that of NCX1 renders the mutant protein insensitive to FK506.