Spontaneously beating sino-atrial node (S-A node) of guinea-pig was superfused with solutions of various osmolarities. The changes in the rate of spontaneous beating and in action potentials were measured. In the solution of normal tonicity, the rate of spontaneous beating was 214 +/- 6 beats/min (mean +/- S.E.) and the maximum rate of rise was 9.7 +/- 2.5 V/s. When extracellular Na+ was reduced by 33%, isotonically replaced with sucrose, the heart rate was reduced to 177 +/- 8 beats/min and the maximum rate of rise was reduced to 7.6 +/- 1.2 V/s. Decreasing the osmolarity by 30% increased the heart rate by 6% and increasing the osmolarity to 130, 150, and 170% decreased the heart rate to 94, 89, and 73%, respectively, in low Na+ medium. The tonicity dependence of the heart rate was not affected by TTX, atropine, phentolamine, or propranolol. When the tonicity was either increased twice or decreased half, the spontaneous beating stopped. Adding either K+ or Rb+ of 6 mM to the solution which was two times as hypertonic as the normal, could activate the beating again. Substances inhibiting the potassium conductance such as tetraethylammonium (TEA), 4-aminopyridine (4-AP), and cesium chloride (CsCl) could also reactivate the spontaneity. Although lowering extracellular Ca2+ concentration could reactivate the spontaneity in the hypertonic medium, increasing Mg2+ inhibited the reactivation. The contribution of K+ current in the effects of changing osmolarity on the spontaneous beating is discussed.