This study was designed to determine whether ouabain could inhibit insulinstimulated ion and/or glucose transport in rat ventricular slices. In the slices which were obtained by tearing with forceps from the inside surface of the ventricle, insulin (0.1-100 ng/ml, 20-min treatment) stimulated ATP hydrolysis (nmol inorganic phosphate/mg tissue/20 min) and ⁸⁶Rb uptake (nmol Rb/mg tissue/8 min) in a concentration-dependent fashion by up to about 25%, compared with control experiment. About 10-fold higher concentrations of insulin stimulated glucose-[¹⁴C] uptake by the slices (nmol glucose/mg tissue/30 min). Both the stimulant effects of insulin on ion and glucose transport were inhibited by ouabain (0.1-1 mM, 20-min treatment) in a similar concentration-response relationship. It thus never appears that two or more independent transmembrane signals would be generated simultaneously in response to the insulin-receptor interaction to operate ion and glucose transport system in rat cardiac muscle. In addition, rat ventricular slices were shown to provide a sensitive tool for the evaluation of insulin effects on ion transport systems in the muscle.