Spontaneously active rabbit sinoatrial node (SAN) cells were bathed in K-free solution or in K-free ouabain (20 microM)-containing solution to depress the electrogenic Na(+)-K+ pump activity. In SAN cells exposed to K-free solution, the automatic action potentials ceased with gradual depolarization, followed by an eventual steady-state membrane potential of -32 +/- 1 mV. Under conditions where the Na(+)-K+ pump was blocked, removal of external Na+ produced a large and rapid hyperpolarization in the membrane potential and the membrane was hyperpolarized by 23 +/- 0.5 mV. When the external Na+ was lowered, Na+ was replaced by Li+. The Na-free hyperpolarization was not affected by applications of verapamil (4 microM), lidocaine (1 mM), and quinidine (50 microM), but was inhibited by either quinacrine (50 microM) or Cd2+ (10 mM), which are blockers of Na(+)-Ca2+ exchange. In the absence of external K+, replacement of external NaCl by sucrose produced a hyperpolarization similar to that seen in the replacement of external Na+ by Li+. In the K-free ouabain (20 microM)-containing solution, removal of external Na+ also produced a hyperpolarization, and the membrane potential dropped from -29 +/- 1 to -48 +/- 1 mV. The intracellular acidification due to NH4Cl removal after exposure to NH4Cl (20 mM) produced a decrease in Na-free hyperpolarization, which in the presence of ouabain was inhibited by the application of Cd2+ (10 mM). Removal of external Ca2+ nearly completely blocked Na-free hyperpolarization. It can be concluded that Na-free hyperpolarizations are related to the functioning of an electrogenic Na(+)-Ca2+ exchange mechanism.