Effect of a Na⁺ gradient on Ca²⁺ uptake was studied in isolated sarcolemmal vesicles of cat ileal longitudinal muscle. Ca²⁺ uptake was markedly stimulated in the presence of an outwardly directed Na⁺ gradient. External Na⁺, monensin and A23187 abolished the Na⁺-dependent Ca²⁺ uptake. Monovalent cations such as K⁺, Li⁺, Rb⁺, Cs⁺ and choline could not substitute for Na⁺ in enhancement of Ca²⁺ uptake. Divalent cations such as Ba²⁺, Sr²⁺, Mn²⁺ and Cd²⁺ but not Mg²⁺ inhibited the Na⁺-dependent Ca²⁺ uptake. Increase in external pH in the range of 6.0 to 8.0 stimulated the Na⁺-dependent Ca²⁺ uptake. Amiloride inhibited the Na⁺-dependent Ca²⁺ uptake at concentrations above 0.5 mM, whereas diltiazem or vanadate did not. The apparent Km of the Na⁺-dependent Ca²⁺ uptake for Ca²⁺ was 18.2 μM and apparent Vmax was 689.7 pmole/mg protein/5 sec. Kinetic analysis of the Na⁺-dependent Ca²⁺ uptake showed a noncompetitive interaction between internal Na⁺ and external Ca²⁺. The dependence of Ca²⁺ uptake on internal Na⁺ showed sigmoidal kinetics and Hill coefficient for internal Na⁺ was 2.52. Inside positive membrane potential generated by imposing an inwardly directed K⁺ gradient and valinomycin significantly stimulated the Na⁺-dependent Ca²⁺ uptake. These results indicate that a Na⁺-Ca²⁺ exchange system exists in the sarcolemmal membranes isolated from cat ileal longitudinal muscle and it might operate as an electrogenic process.