Cardiomyopathy associated mutations in troponin often affect the actin-activated ATPase activity of myosin and the response of force and ATPase activity to Ca2+. Three hypertrophic cardiomyopathy TnC mutants A8V, C84Y, and D145E were examined for their effects on the distribution of states of regulated actin in vitro. In the absence of activating Ca2+, regulated actin filaments containing the C84Y mutant produced a 2.5x elevation in ATPase activity of skeletal myosin subfragment 1 compared to wild type; the other mutants had normal activity. We examined acrylodan-tropomyosin fluorescence changes that occur following rapid detachment of S1 from regulated actin in the absence of Ca2+. All troponin mutants gave a fluorescence increase indicative of a normal transition into the inactive state. The A8V mutant differed from wild type and other TnC mutants at saturating Ca2+. Actin filaments containing the A8V TnC mutant produced an ATPase rate in Ca2+ that was 1.3x wild type. For comparison, the Δ14TnT mutant that destabilizes the inactive state was 1.7x wild type. Interestingly, the effects of the A8V-TnC and Δ14-TnT mutants were additive leading to a Ca2+-activated rate 2.3x wild type. The A8V TnC mutant appeared to favor the active state relative to wild type but only in the presence of Ca2+. This behavior differed from the Δ14 mutation of TnT where the distribution was also altered at low Ca2+ conditions.