Data from intact and skinned muscle fibers support the hypothesis that cross-bridge interaction modifies TnC structure and calcium activation. Barnacle single muscle fibers microinjected with the calcium bioluminescent photoprotein, aequorin, show extra light (calcium) when shortened during the declining phase of the calcium transient. The extra calcium is increased by increases in muscle force, and its decline is delayed at higher forces. This extra calcium occurs probably because calcium binding to the activating sites is increased by cross-bridge interaction. In rabbit muscle, TnC structure is modified by cross-bridge interaction, since in skinned rabbit psoas muscle fibers TnC extraction is slower at shorter sarcomere lengths, where cross-bridge attachment is increased. Thus the rigor bridges formed in the extraction solution strengthen the attachment of TnC to the thin filament. Reintroduction of TnC, labeled with fluorescent probes near the Ca specific binding sites (Danzylaziridine-DANZ) and Ca-Mg sites (Rhodamine), into the partially TnC extracted fibers allows us to assess the structural changes (total fluorescence for the DANZ probes, linear dichroism for the RHOD probe) in response to calcium binding and cross-bridge attachment. At sarcomere lengths beyond overlap, calcium binding increases the DANZ-TnC fluorescence and disorders the RHOD-TnC label. At full overlap of filaments, rigor cross-bridges also increase the DANZ-TnC fluorescence and RHOD-TnC disorder. The addition of calcium in rigor increases the DANZ-TnC fluorescence little but causes additional RHOD-TnC disorder, although both fluorescence and disorder are increased further in the presence of calcium plus MgATP. In fibers containing DANZ-TnC, decreasing MgATP in the absence of calcium increases both the force and the fluorescence as rigor cross-bridges activate the muscle. In the presence of calcium, an increase in MgATP to 0.75 microM produces a small fluorescent enhancement, but an increase in MgATP to 10 microM and to 3 mM produces a substantial enhancement. The data imply that calcium activates the thin filament, but that the filament is activated further by rigor cross-bridges. Active cross-bridges activate the thin filament still further. Thus, cross-bridges modify TnC structure and calcium activation, with active cross-bridges being more effective than rigor cross-bridges.