—To investigate the kinetic parameters of the crossbridge cycle that regulate force and shortening in cardiac muscle, we compared the mechanical properties of cardiac trabeculae with either ATP or 2-deoxy-ATP (dATP) as the substrate for contraction. Comparisons were made in trabeculae from untreated rats (predominantly V1 myosin) and those treated with propylthiouracil (PTU; V3 myosin). Steady-state hydrolytic activity of cardiac heavy meromyosin (HMM) showed that PTU treatment resulted in >40% reduction of ATPase activity. dATPase activity was >50% elevated above ATPase activity in HMM from both untreated and PTU-treated rats. V_max of actin-activated hydrolytic activity was also >50% greater with dATP, whereas the K_m for dATP was similar to that for ATP. This indicates that dATP increased the rate of crossbridge cycling in cardiac muscle. Increases in hydrolytic activity were paralleled by increases of 30% to 80% in isometric force (F_max), rate of tension redevelopment (k_tr), and unloaded shortening velocity (V_u) in trabeculae from both untreated and PTU-treated rats (at maximal Ca²⁺ activation), and F-actin sliding speed in an in vitro motility assay (V_f). These results contrast with the effect of dATP in rabbit psoas and soleus fibers, where F_max is unchanged even though k_tr, V_u, and V_f are increased. The substantial enhancement of mechanical performance with dATP in cardiac muscle suggests that it may be a better substrate for contractility than ATP and warrants exploration of ribonucleotide reductase as a target for therapy in heart failure.