Cardiac hypertrophy induced by thyrotoxic stress leads to an increase in the rate of force development, velocity of shortening, tension-dependent heat generation, and myosin ATPase activity. We did studies to see whether alterations in covalent phosphorylation of myofibrillar proteins correlate with these changes. The protein preparations were isolated from control and thyrotoxic hearts of male albino rabbits freeze-clamped in situ. We measured myofibrillar ATPase, and the covalent phosphate content of ventricular myosin 19,000 (mol wt) light chain (P-light chain) and troponin I (TnI). The myofibrillar ATPase activity was increased 2-fold in the thyrotoxic preparations with no change in the level of myofibrillar phosphorylation. The covalent phosphate content of TnI was 1.21 +/- 0.09 mol P/mol TnI in control hearts and 1.14 +/- 0.04 mol P/mol TnI in thyrotoxic hearts. The covalent phosphate content of the light chain fraction was 0.41 +/- 0.06 mol P/mol P-light chain in control hearts and 0.37 +/- 0.04 mol P/mol P-light chain in thyrotoxic hearts. The dependence of the normalized myofibrillar ATPase on free calcium concentration was the same in control and thyrotoxic preparations. Thus the mechanical, thermal, and biochemical changes found in hearts from thyrotoxic animals probably occur with no change in phosphorylation of TnI or myosin light chains.