The effects of Ca2+ and myosin subfragment 1 on the conformation of tropomyosin and actin in regulated actin filaments in ghost fibers were investigated by means of the polarized fluorescence technique. Regulated thin filaments were reconstituted in skeletal muscle ghost fibers by incorporation into the fibers of either skeletal muscle troponin-tropomyosin or smooth-muscle caldesmon-calmodulin-tropomyosin complexes. Tropomyosin and actin were specifically labeled with fluorescent probes, 1,5-IAEDANS and phalloidin-rhodamine, respectively. Analysis of the fluorescence parameters indicated that the binding of Ca2+ to regulated actin filaments induces conformational changes in tropomyosin and actin that lead to the strengthening of the interaction between these two proteins and weakening of the binding of actin monomers in the filament. These changes become larger when regulated actin forms rigor links with myosin subfragment 1. No notable alterations in the position of tropomyosin relative to actin in the frontal plane of the fiber were detected either upon binding of Ca2+ or upon the additional binding of myosin subfragment 1 to regulated actin.