Calmodulin (CaM) and troponin C (TnC) are evolutionarily and structurally homologous, yet they are not functionally interchangeable. In particular, CaM cannot effectively substitute for TnC as an activator of skeletal muscle contraction. To determine if this is a consequence of CaM's weak association with troponin T and I or the result of a more fundamental mechanistic defect, we have used CaM and a CaM[TnC] chimera, CaM[3,4 TnC], that stably associates with the thin filament. Replacement of TnC with CaM or CaM[3,4 TnC] reveals that CaM-like molecules reduce the Ca(2+)-sensitivity and cooperativity of activation, as well as the maximal Ca(2+)-activated tension. These observations indicate that CaM-like molecules are unable to continuously maintain the activated state of the thin filament.