The effects of the removal of fast skeletal troponin C (fsTnC) and its replacement by cardiac troponin C (cTnC) and the exchange of fast skeletal troponin (fsTn) for cardiac troponin (cTn) were measured in rabbit fast skeletal myofibrils. Electrophoretic analysis of myofibril suspensions indicated that replacement of fsTnC or exchange of fsTn with cTnC or cTn respectively was about 90% complete in the protocols used. Mechanical measurements in single myofibrils, maximally activated by fast solution switching, showed that replacement of fsTnC by cTnC reduced the isometric tension, the rate of tension rise following a step increase in calcium (k_ACT), and the rate of tension redevelopment following a quick release and restretch (k_TR), but had no effect on the kinetics of the fall in tension when the concentration of inorganic phosphate (P_i) was abruptly increased (k_Pi(+)). These data suggest that the chimeric protein produced by cTnC replacement in fsTn altered those steps controlling the weak to strong cross-bridge attachment transition. Inefficient signalling within the chimeric troponin may cause these changes. However, replacement of fsTn by cTn by exchange had no effect on maximal isometric tension, k_ACT, or k_TR, suggesting that these mechanics are largely determined by the isoform of the myosin molecule. Replacement of fTn by cTn, on the other hand, shifted the pCa₅₀ of the pCa-tension relationship from 5.70 to 6.44 and reduced the Hill coefficient from 3.3 to 1.4, suggesting that regulatory protein isoforms primarily alter calcium sensitivity and cooperativity of the force generating mechanism.