In this study, the effects of carbachol (CCh) on twitch tension, intracellular Na<sup>+</sup> activity (a<sup>i</sup><sub>Na</sub>), and action potential were simultaneously measured in canine cardiac Purkinje fibers in order to examine the regulation of inotropy through muscarinic receptors and its relation to a<sup>i</sup><sub>Na</sub>. In fibers driven at 1 Hz, CCh (10 μM) initially and transiently decreased and then increased the twitch tension by 36 ± 8%. The action potential showed a significant elevation of the plateau and a significant shortening of the duration at 90% repolarization (APD<sub>90</sub>), from 403 ± 7 to 389 ± 7 ms. The a<sup>i</sup><sub>Na</sub> decreased from 7.4 ± 0.4 to 6.7 ± 0.3 mM (n = 23, p < 0.05). Atropine (1 μM) decreased the twitch tension by 21 ± 6% (n = 7, p < 0.05) without significant effects on the action potential and a<sup>i</sup><sub>Na</sub>, and inhibited the effects of CCh. Cs<sup>+</sup> (20 mM) increased the plateau height and APD<sub>90</sub>, enhanced the twitch tension by 66 ± 24%, but decreased a<sup>i</sup><sub>Na</sub> from 7.3 ± 0.3 to 6.3 ± 0.4 mM (n = 6, p < 0.05). In the presence of 20 mM Cs<sup>+</sup>, some fibers generated slow responses. The addition of 10 μM CCh further increased the twitch tension and APD<sub>90</sub>, and decreased a<sup>i</sup><sub>Na</sub> from 6.3 ± 0.4 to 5.3 ± 0.3 mM. Ouabain (0.3 μM) increased the twitch tension and a<sup>i</sup><sub>Na</sub>, and inhibited the CCh-induced decrease of a<sup>i</sup><sub>Na</sub>. In the presence of ouabain, 20 mM Cs<sup>+</sup> depolarized the fiber and generated slow responses with a decreased a<sup>i</sup><sub>Na</sub>. The addition of 10 μM CCh enhanced the slow action potential, and increased a<sup>i</sup><sub>Na</sub> although there was a transient decrease during early exposure. These results suggest that activation of muscarinic receptors in canine Purkinje fibers results in an enhancement of the Na<sup>+</sup>-K<sup>+</sup> pump activity and a biphasic inotropic response, probably via different receptor subtypes. The inhibitory effect, most likely through M<sub>2</sub> receptors, is associated with the activation of K<sup>+</sup> channels. The stimulatory effect, on the other hand, is probably due to the action on the M<sub>1</sub> receptors, resulting in increases in Ca<sup>2+</sup> currents.