We have investigated the effect of caldesmon on the actin conformational state and its position at force generation in glycerinated fibers upon transformation from relaxation to rigor. F-actin and caldesmon were labeled with TRITC-phalloidin or acrylodan, respectively, and the orientation and mobility of the probes were calculated. Transition from relaxation to rigor was accompanied by force development and by the changes in orientation and mobility of TRITC-phalloidin that were typical for actin monomer transformation from the “OFF” to the “ON” conformational state. In the presence of caldesmon, both the force developed by the fibers and the changes in the orientation and mobility of TRITC-phalloidin were markedly decreased. In contrast, the orientation and mobility of acrylodan change essentially showed the displacement of the caldesmon molecules and the changes in its mobility. The results are evidence that structure and/or mode of the attachment of caldesmon to actin modulates both the force production and transition of actin monomers from “OFF” to “ON” conformations in the ATPase cycle.