Na+-Ca2+ exchange (NCX) current has been suggested to play a role in cardiac pacemaking, particularly in association with Ca2+ release from the sarcoplasmic reticulum (SR) that occurs just before the action potential upstroke. The present experiments explore in more detail the contribution of NCX to pacemaking. Na+-Ca2+ exchange current was inhibited by rapid switch to low-Na+ solution (with Li+ replacing Na+) within the time course of a single cardiac cycle to avoid slow secondary effects. Rapid switch to low-Na+ solution caused immediate cessation of spontaneous action potentials. ZD7288 (3 microM), to block I(f) (funny current) channels, slowed but did not stop the spontaneous activity, and tetrodotoxin (10 microM), to block Na+ channels, had little effect, but in the presence of either of these agents, rapid switch to low-Na+ solution again caused immediate cessation of spontaneous action potentials. Spontaneous electrical activity was also stopped following loading of the cells with the Ca2+ chelators BAPTA and EGTA, and by exposure to the NCX inhibitor KB-R7943 (5 microM). When rapid switch to low-Na+ solution caused cessation of spontaneous activity, this was found (using confocal microscopy, with fluo-4 as the Ca2+ probe) to be accompanied by an initial fall in cytosolic [Ca2+], with subsequent appearance of Ca2+ waves. Inhibition of SR Ca2+ uptake with cyclopiazonic acid (CPA, 30 microM) slowed but did not stop spontaneous activity. Rapid switch to low-Na+ solution in the presence of CPA caused abolition of spontaneous Ca2+ transients and a progressive rise in cytosolic [Ca2+]. With ratiometric fluorescence methods (indo-5F as the Ca2+ probe), the minimum level of [Ca2+] between beats was found to be approximately 225 nM, and abolition of beating with nifedipine, acetylcholine or adenosine caused a fall in cytosolic [Ca2+] below this level. These observations support the hypothesis that NCX current is essential for normal pacemaker activity under the conditions of our experiments. A continuous depolarizing influence of current through the NCX protein might result from maintained electrogenic NCX (with 3:1 stoichiometry, supported by a cytosolic [Ca2+] that normally does not fall below 225 nM between beats) and/or from a novel, recently suggested role of the NCX protein to allow a Na+ leak pathway.