The kinetic properties of the myofibrillar system of atrial and ventricular myocardia of hyperthyroid rats were analyzed by determining ATPase activity and maximum shortening velocity. Hyperthyroidism was induced by daily subcutaneous injections of triiodothyronine (0.2 mg/kg body wt) for 2 wk. The treatment induced a marked atrial and ventricular hypertrophy and, in ventricular myocardium, an isomyosin shift toward a homogeneous V1 composition. Skinned trabeculae and purified myofibrils were prepared from atrial and ventricular myocardia. Enzymatic assays on the myofibrils showed that both Ca-stimulated ATPase activity and Ca-Mg-dependent ATPase activity had equal values in atrial and ventricular myocardia. In skinned trabeculae during maximal Ca activations, force-velocity curves were determined by load-clamp maneuvers, and unloaded shortening velocity (Vo) was obtained with the slack-test method. Both maximum shortening velocities extrapolated from the force-velocity curves (Vmax) and Vo were significantly higher (+68 and +52%, respectively) in atrial than in ventricular preparations. Developed tension was significantly greater in ventricular preparations. Maximum power output was not significantly different. Previous findings (V. Cappelli, R. Bottinelli, C. Poggesi, R. Moggio, and C. Reggiani. Circ. Res. 65: 446-457, 1989) had led to the conclusion that variations in ATPase activity and shortening velocity of ventricular myocardium can be accounted for by changes in isomyosin composition. In this light, the present results suggest that 1) ATPase activity is equal in atrial and ventricular myocardia as the two tissues contain the same myosin heavy chain isoform, 2) the difference in maximum speed of shortening between atrium and ventricle might be due to the presence of tissue-specific isoforms of myosin light chains.