Using a primary culture of neonatal rat heart, we investigated the properties of iodothyronine 5'-deiodinating activity in the heart. I-125-release from [I-125] reverse T3 incubated with cell homogenates depended on the concentrations of protein and dithiothreitol, pH, incubation time, and temperature of the incubation mixture. The activity was almost completely inhibited by 10(-3)-10(-5) M propylthiouracil and had the higher affinity for reverse T3 than T4. These findings indicate that type I iodothyronine 5'-deiodinase(I-5'-D) exists in cultured neonatal rat myocardial cells. Then, we studied the direct effects of T3, insulin, glucocorticoid, and angiotensin II (AII) on myocardial I-5'-D. The addition of 10(-9)-10(-7) M T3 or 10(-9)-10(-6) M AII to the culture medium increased I-5'-D activity in a dose-dependent manner. Insulin and dexamethasone, however, had no significant effects. Kinetic studies revealed that maximum velocities in T3- and AII-treated cells were 2.1- and 1.8-fold above the control value, respectively, whereas the apparent Michaelis-Menten constant did not alter significantly both in T3- and AII-treated cells. Moreover, the treatment of cyclohexamide combined with T3 or AII completely abolished the stimulating effect of these agents on I-5'-D activity. From these data, it is suggested that both T3 and AII increase the activity of myocardial I-5'-D by their direct actions to the heart, probably through the new synthesis of I-5'-D.