The effect of troponin T treatment on the Ca(2+)-activated tension of single glycerinated rabbit skeletal muscle fibers was examined. The tension of the fiber was completely desensitized to Ca2+ by incubation in a solution containing an excessive amount of troponin T and reached a level of about 70% of the maximum tension of the control fiber. SDS/PAGE showed that most of troponins C and I was removed from the fiber by troponin T treatment. During the course of troponin T treatment, the cooperativity of Ca2+ activation (Hill coefficient) was decreased while pCa at half-maximal Ca(2+)-sensitive tension (pK) increased. Using the 26-K fragment of troponin T, the study indicated that the removal of troponins C and I was due to the replacement of the troponin C.I.T complex in the myofibrils of the fiber with the added troponin T. The troponin-T-treated fiber was again sensitized to Ca2+ by the addition of troponin C.I. The removal of troponin C by treatment with trans-1,2-cyclohexanediamine-N,N,N',N'-tetraacetic acid did not change the minimum tension of the fiber, from which troponin C.I was partially removed by troponin T treatment, but it decreased the height of maximum tension with a concomitant decrease in the Hill coefficient as well as a decrease in pK. The above findings suggested that pK is determined by the balance between two opposite actions through troponins C and I, while the extent of cooperativity of Ca2+ activation seemed to be related mainly to the content of troponin C.