The Na(+)-Ca(2+) exchanger (NCX) is a major Ca(2+) regulating protein. It is almost ubiquitously expressed. Cyclophilins (Cyps) make up a class of proteins that are involved in protein folding via their peptidyl prolyl cis-trans isomerase (PPIase) and chaperone domains. They are also the cellular receptors of cyclosporin A (CsA). Binding of CsA to cyclophilins inhibits both PPIase and chaperone activities. We have shown that treatment of transfected HEK 293 cells expressing the Na(+)-Ca(2+) exchanger NCX1 with CsA results in downregulation of surface expression and transport activity, without any reduction in the total level of cell NCX1 protein [Kimchi-Sarfaty, C., et al. (2002) J. Biol. Chem. 277 (4), 2505-2510]. In this work, we show that knockdown of cell CypA using targeting siRNA (without any CsA treatment) results in a reduction in the level of NCX1 surface expression, a decrease in the level of Na(+)-dependent Ca(2+) uptake, and no change in the total amount of cell NCX1 protein in NCX1.5-transfected HEK 293 cells and nontransfected H9c2 cells that express NCX1.1 naturally. It also reduced Na(+)-dependent Ca(2+) fluxes measured by changes in Fluo-4 AM fluorescence in single NCX1.5-transfected HEK 293 and single H9c2 cells. Knockdown of CypB had no significant effect on either transport activity, surface expression, NCX1 cell protein expression, or Ca(2+) fluxes. Overexpression of CypA or its R55A mutant, which exhibits a substantially reduced PPIase activity, alleviated the reduction of NCX1 surface expression caused by CsA treatment, suggesting that the PPIase domain was probably not mandatory for NCX1 functional expression. We suggest that CypA plays a role in the functional expression of NCX1 protein.