The Na+-Ca2+ exchange was studied in a highly purified vesicular preparation derived from heart sarcolemma. The initial velocity of the Na+-driven Ca2+ influx, which was monitored continuously with a specific electrode, was 15 nmol/mg of protein per sec; the total Ca2+-accumulation capacity was 80 nmol/mg of protein. The Na+-Ca2+ exchange generated a current that was compensated for by the uptake of tetraphenylphosphonium in (Ph4P+) (when the latter was present in the medium), the influx of K+, and the efflux of Cl-. The movements of Ph4P were followed with a specific electrode. Ca2+ in the concentration range 3-50 microM induced an increase in the permeability of the sarcolemmal membrane to K+. Under conditions of optimal charge neutralization by K+ (i.e., in the presence of valinomycin), the Km (Ca2+) of the exchanger was 1.5 microM. The Na+-Ca2+ exchange was inhibited by chlorpromazine and was not inhibited by vanadate.