Effects of isoprenaline, carbachol, and Cs+ on intracellular Na+ activity (a(i)Na) and spontaneous action potentials were studied in multicellular and single cell preparations isolated from rabbit sinoatrial (SA) nodes. a(i)Na was measured with double-barreled Na+-selective microelectrodes and the fluorescent Na+-indicator sodium-binding benzofuran isophthalate (SBFI). In spontaneously beating cells, aiNa measured with Na+-selective microelectrodes and SBFI were 4.5 +/- 1.2 mM (means +/- SD, n = 21) in multicellular preparations and 4.0 +/- 1.1 mM (n = 16) in single cells, respectively. Measurements of a(i)Na with microelectrodes showed that isoprenaline increased a(i)Na from 4.7 +/- 1.2 to 5.5 +/- 1.6 mM (n = 16, P < 0.01) and shortened the action potential cycle length (ACL) from 338 +/- 46 to 269 +/- 35 ms (n = 16, P < 0.01). However, increasing the action potential rate by pacing produced a much smaller increase in a(i)Na. Changes in a(i)Na and ACL produced by isoprenaline were blocked by Cs+. The selective hyperpolarization-activated inward current (If) blocker ZD-7288 decreased a(i)Na from 5.2 +/- 1.0 to 4.6 +/- 1.3 mM (n = 4, P < 0.01) and prolonged ACL from 394 +/- 20 to 553 +/- 68 ms (n = 4, P < 0.01). The If blocker substantially inhibited the increase in a(i)Na produced by isoprenaline. Carbachol and Cs+ decreased aiNa from 4.6 +/- 1.4 to 3.9 +/- 1.2 mM (n = 15, P < 0.01) and from 4.9 +/- 1.0 to 3.9 +/- 1.3 mM (n = 18, P < 0.01), respectively. In addition, carbachol and Cs+ prolonged ACL from 345 +/- 44 to 587 +/- 100 ms (n = 15, P < 0.01) and from 353 +/- 30 to 464 +/- 87 ms (n = 18, P < 0.01), respectively. However, carbachol and Cs+ almost did not change a(i)Na when SA node cells became quiescent in a 25.4 mM extracellular K+ concentration. The results suggest that isoprenaline, ZD-7288, carbachol, or Cs+ might have changed a(i)Na and action potential rate by possibly stimulating or inhibiting If carried by Na+. Measurements of a(i)Na with SBFI showed that isoprenaline, carbachol, and Cs+ produced a(i)Na changes that were similar to those measured with the microelectrodes.