1. Two-dimensional electrophoresis has been used to study the extent of phosphorylation of the P light chain of myosin and troponin I in the rabbit beating heart. 2. A procedure has been developed that eliminates endogenous protein phosphatase activity during homogenization and sample preparation for electrophoresis. 3. Evidence has been obtained for two unphosphorylated forms of the P light chain in myosin from the ventricle of the rabbit, guinea pig and cow. 4. In vivo and in the rabbit perfused beating heart about 25% of the P light-chain fraction is in the phosphorylated form. 5. Intervention with adrenaline produced a slight increase in the extent of phosphorylation that reached a maximum after the peak in inotropic response. A similar increase was obtained with ischaemia in the absence of adrenaline. 6. The changes in phosphorylation of the major forms of troponin I identified by electrophoresis occurred after the peak of response to adrenaline and were compatible with previous results.
