The effects of α₁-adrenergic receptor stimulation on membrane potential, intracellular Na⁺ activity, and twitch force were investigated in ventricular muscles from guinea-pig hearts. Action potentials, intracellular Na⁺ activity, and twitch force of ventricular papillary muscles were measured simultaneously under various experimental conditions. Stimulation of the α₁-adrenergic receptor by phenylephrine produced variable changes in action potential duration, a slight hyperpolarization of the diastolic membrane potential, a decrease in intracellular Na⁺ activity, and a biphasic inotropic response in which a transient negative inotropic response was followed by a sustained positive inotropic response. These changes were blocked by prazosin, an antagonist of the α₁-adrenergic receptor, but not by atenolol, an antagonist of the β-adrenergic receptor. This indicates that the changes in membrane potential, intracellular Na⁺ activity, and twitch force are mediated by stimulation of the α₁-adrenergic receptor, but not by stimulation of β-adrenergic receptor. The decrease in intracellular Na⁺ activity was not observed in quiescent muscles, depending on the rate of the action pontentials in beating muscles. The intracellular Na⁺ activity decrease was substantially inhibited by tetrodotoxin. However, the decrease in intracellular Na⁺ activity was not affected by an inhibition of the Na⁺-K⁺ pump. Therefore, the decrease in intracellular Na⁺ activity mediated by the α₁-adrenergic receptor appears to be due to a reduction of Na⁺ influx during the action potential, perhaps through tetrodotoxin sensitive Na⁺ channels. Our study also revealed that the decrease in intracellular Na⁺ activity might be related to the transient negative inotropic response. The intracellular Na⁺ activity decrease could lower intracellular Ca²⁺ through the Na⁺-Ca²⁺ exchanger and thereby produce a decline in twitch force.