We find that extraction of as little as one troponin C molecule per troponin-tropomyosin strand on a thin filament reduces the slope of the pCa/tension relation. We interpret this to mean that the regulatory units along a thin filament of rabbit psoas fibers are linked co-operatively so that a thin filament activates as a unit. The presence of extended co-operativity explains why the pCa/tension relation in skinned fibers has a slope much higher than predicted by binding of Ca²⁺ to one regulatory unit. Replacement of the extracted troponin C with purified troponin C fully reverses the effect of extraction and shows it to be the essential Ca²⁺ binding protein responsible for the steep slope of the pCa/tension relation.