Changes in skeletal troponin C (sTnC) structure during thin filament activation by Ca 2+ and strongly bound cross-bridge states were monitored by measuring the linear dichroism of the 5′ isomer of iodoacetamidotetramethylrhodamine (5′IATR), attached to Cys 98 (sTnC-5′ATR), in sTnC-5′ATR reconstituted single skinned fibers from rabbit psoas muscle. To isolate the effects of Ca 2+ and cross-bridge binding on sTnC structure, maximum Ca 2+-activated force was inhibited with 0.5 mM AlF 4 − or with 30 mM 2,3 butanedione-monoxime (BDM) during measurements of the Ca 2+ dependence of force and dichroism. Dichroism was 0.08 ± 0.01 (± SEM, n = 9) in relaxing solution (pCa 9.2) and decreased to 0.004 ± 0.002 (± SEM, n = 9) at pCa 4.0. Force and dichroism had similar Ca 2+ sensitivities. Force inhibition with BDM caused no change in the amplitude and Ca 2+ sensitivity of dichroism. Similarly, inhibition of force at pCa 4.0 with 0.5 mM AlF 4 − decreased force to 0.04 ± 0.01 of maximum (± SEM, n = 3), and dichroism was 0.04 ± 0.03 (± SEM, n = 3) of the value at pCa 9.2 and unchanged relative to the corresponding normalized value at pCa 4.0 (0.11 ± 0.05, ± SEM; n = 3). Inhibition of force with AlF 4 − also had no effect when sTnC structure was monitored by labeling with either 5-dimethylamino-1-napthalenylsulfonylaziridine (DANZ) or 4-( N-(iodoacetoxy)ethyl- N-methyl)amino-7-nitrobenz-2-oxa-1,3-diazole (NBD). Increasing sarcomere length from 2.5 to 3.6 μm caused force (pCa 4.0) to decrease, but had no effect on dichroism. In contrast, rigor cross-bridge attachment caused dichroism at pCa 9.2 to decrease to 0.56 ± 0.03 (± SEM, n = 5) of the value at pCa 9.2, and force was 0.51 ± 0.04 (± SEM, n = 6) of pCa 4.0 control. At pCa 4.0 in rigor, dichroism decreased further to 0.19 ± 0.03 (± SEM, n = 6), slightly above the pCa 4.0 control level; force was 0.66 ± 0.04 of pCa 4.0 control. These results indicate that cross-bridge binding in the rigor state alters sTnC structure, whereas cycling cross-bridges have little influence at either submaximum or maximum activating [Ca 2+].