The relation between single skinned skeletal fiber contractile mechanics and the myosin mechanoenzyme was examined by perturbing the actomyosin interaction with the ATP analog CTP in fibers from both rabbit psoas and rat soleus. Tension, instantaneous stiffness, and the rate of tension redevelopment (ktr), under software-based sarcomere length control, were examined at 15 degrees C for a range of Ca2+ concentrations in both fiber types. CTP produced 94% of the maximum ATP-generated tension in psoas fibers and 77% in soleus fibers. In psoas, CTP also increased stiffness to 106% of the ATP stiffness, whereas in soleus stiffness decreased to 92%. Thus, part of the greater difference between maximum ATP- and CTP-generated tension in soleus fibers appears to be due to a decrease in strongly bound cross-bridge number. Interestingly, although the nucleotide exchange produced substantial increases in the steepness (nH) of the tension- and stiffness-pCa relationships in soleus fibers, only minor changes were seen in psoas fibers. At maximum Ca2+ and nominal P(i) levels, ktr in psoas fibers increased from 11.7 s-1 with ATP to 16.6 s-1 with CTP and in soleus fibers from 4.9 to 8.4 s-1. Increased P(i) levels decreased the maximum Ca(2+)-activated tension in both fiber types and increased the ktr of psoas fibers, but the ktr of soleus fibers was not significantly altered. Thus, although the nucleotide exchange generally produced similar changes in the mechanics, there were significant muscle lineage differences in the tension- and stiffness-pCa relations and in the effects of P(i) on ktr, such that differences in contractile mechanics were lessened in the presence of CTP.