The total ATPase activity of myosin and the values for the isozyme V, have been measured in hearts from rats of different ages and with different levels of thyroid function. The contribution of V3was calculated from the difference between total and V1ATPase, neglecting the small contribution of V2. Hearts were quickly frozen after rapid removal from the animals in order to preserve the state of ATPase activity that existed in the intact animal, and ATPase activity was measured in thin sections of tissue by a microphotometric technique. In euthyroid hearts, although cAMP increases total myosin ATPase activity and the activity of V1, the cyclic nucleotide inhibits the ATPase activity of V,. In hearts from rats with developing hypothyroidism following thyroidectomy, the same occurs. After a sufficient period has elapsed after thyroidectomy for V1to have practically disappeared, cAMP has no effect on ATPase activity, but the injection of thyroid hormone restores the effect. Total myosin ATPase activity is maintained relatively constant as the animal ages from 80 to 165 days and during the first 10–11 days following thyroidectomy even though the concentration of V1is dropping. The explanation proposed for these observations is that myosin can exist in two different forms, only one of which can participate in the active generation of force. The transition between the two forms is regulated by a soluble factor that is itself controlled by the adrenergic system. The factor(s) involved in this regulatory mechanism is soluble and can be transferred between different thin sections cut from a frozen heart.