Calcium regulation of vertebrate striated muscle contraction is initiated by conformational changes in the Ca2+-binding protein troponin C (TnC) and subsequent changes in the interaction of TnC with the inhibitory protein TnI and the tropomyosin-binding protein TnT. We have used the frequency domain method of fluorescence resonance energy transfer to measure TnT-TnC, TnT-TnI and TnI-TnC distances and distributions, in the presence of Ca2+, Mg2+, or EGTA, in TnC-TnI-TnT and TnC-TnI complexes. We reconstituted functional, ternary troponin complexes using the following recombinant subunits whose sequences were based on those of rabbit skeletal muscle: wild-type TnC; {dollar}\rm TnT\sb{lcub}25{rcub},{dollar} a mutant C-terminal 25-kDa fragment of TnT containing a single Trp-212 which was used as the sole donor for fluorescence energy transfer measurements; Trp-less TnI mutants which contained either no Cys or a single Cys at position 9, 96, or 117. The binary Tn complex (TnI-TnC) was reconstituted by using wild-type TnC and mutant TnI which contained no Cys and a single Trp at position 106. Energy acceptor groups were introduced into TnC or TnI by labeling with dansyl aziridine or N-(iodoacetyl)-{dollar}\rm N\sp\prime{dollar}-(1-sulfo-5-naphthyl)ethylenediamine. Our results indicate that the troponin complex is relatively rigid in relaxed muscle, but becomes much more flexible when Ca2+ binds to regulatory sites in TnC. This increased flexibility may be propagated to the whole thin filament, releasing the inhibition of actomyosin ATPase activity and allowing the muscle to contract.