Effects of isoprenaline, carbachol, and Cs⁺ on intracellular Na⁺ activity (aⁱ_Na) and spontaneous action potentials were studied in multicellular and single cell preparations isolated from rabbit sinoatrial (SA) nodes. aⁱ_Na was measured with double-barreled Na⁺-selective microelectrodes and the fluorescent Na⁺-indicator sodium-binding benzofuran isophthalate (SBFI). In spontaneously beating cells, aⁱ_Na 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 aⁱ_Na with microelectrodes showed that isoprenaline increased aⁱ_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 aⁱ_Na. Changes in aⁱ_Na and ACL produced by isoprenaline were blocked by Cs⁺. The selective hyperpolarization-activated inward current (I_f) blocker ZD-7288 decreased aⁱ_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 I_f blocker substantially inhibited the increase in aⁱ_Na produced by isoprenaline. Carbachol and Cs⁺ decreased aⁱ_Na 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 aⁱ_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 aⁱ_Na and action potential rate by possibly stimulating or inhibiting I_f carried by Na⁺. Measurements of aⁱ_Na with SBFI showed that isoprenaline, carbachol, and Cs⁺ produced aⁱ_Na changes that were similar to those measured with the microelectrodes.