T he extent of inhibition of monovalent cation active transport in Purkinje fibers and myocardium in response to toxic and inotropic doses of digitalis were studied in the dog to elucidate the factors underlying the different relative sensitivities of these tissues to the toxic arrhythmogenic effects of digitalis. Monovalent cation transport inhibition was assessed by measuring uptake of the K⁺ analog Rb⁺ in samples of myocardium and Purkinje fibers after in vitro ouabain exposure and after acute or chronic administration of digoxin in vivo. The active uptake of Rb+ was determined as the difference between total uptake and uptake in the presence of 1.0 mM ouabain. Mean active uptake of Rb⁺ by Purkinje fibers from control hearts was 1.62±0.11 (SEM) nmol/mg wet wt per 15 min, significantly greater than the value of 0.49±0.05 for myocardium (P < 0.001). Concentration-effect curves for inhibition of monovalent cation active transport by in vitro exposure to graded concentrations of ouabain showed that the concentration for half-maximal inhibition of monovalent cation transport, IC₅₀, for Purkinje fiber transport was 0.4 μM, significantly less than the value of 1.4 μM for myocardial samples. Dogs were given toxic doses of digoxin (0.3 mg/kg i.v.). At onset of sustained ventricular tachycardia, they were killed and monovalent cation transport measured in myocardial and Purkinje fiber samples. Active Rb⁺ uptake was inhibited by 44% in myocardial samples, whereas a significantly greater inhibition of 76% was noted in Purkinje fibers (P < 0.01). Similar data were obtained for both transmural myocardial biopsy samples and subendocardial trabecular samples obtained from regions adjacent to Purkinje fibers. Another group of dogs received a nontoxic dose of 0.02 mg/kg i.v. daily for 6 d. Myocardium showed a 17% reduction in Rb⁺ active uptake compared to control animals receiving no drug, whereas Purkinje fiber transport was reduced in these dogs to a significantly greater extent averaging 44% (P < 0.001). Thus, both toxic and inotropic (non-toxic) doses of digoxin inhibited monovalent cation transport in Purkinje fibers to a greater extent than in myocardium. This difference in apparent sensitivity of monovalent cation transport to digoxin may contribute to the previously reported tendency of digitalis toxic arrhythmias to arise in Purkinje fibers.