The effects of oxytocin on the beating properties of rat myocardial cells in cultures supplemented with serum, and on the spreading functions in serum-free culture were investigated. Oxytocin significantly increased the beating rates of single cells and cell clusters, and made quiescent single cells beat in a 2-day culture with Eagle's minimum essential medium supplemented with 10% bovine serum. The spontaneous activity of myocardial cells increased by oxytocin was not inhibited by the addition of propranolol, contrary to the case with epinephrine. Oxytocin improved the arrhythmia of myocardial cells which showed partial or continuous cellular fibrillation and mild irregular beating induced by lowering the potassium concentration of culture media. As for the spreading functions, oxytocin increased myosin ATPase activity and protein synthesis, increasing the spreading phenomenon, but did not affect the amounts of ATP and phosphocreatine, or the nucleic acid synthesis of myocardial cells cultured with the sample in serum-free medium for 2 days.