Cardiac troponin I (CTnI) can be phosphorylated by a c-AMP dependent protein kinase. We have investigated the effect of the phosphorylation on the emission decay properties of its single tryptophan by using a cavity dumped and synchronously pumped dye laser system. At 20°C, τ1~0.60 ns, τ2~2.22 ns, and τ3~4.72 ns. The corresponding fluorescence contributions were 7%, 47%, and 46%, respectively. Upon phosphorylation four lifetimes were observed: τ1~0.11 ns, τ2~0.81 ns, τ3~1.95 ns, and τ4~6.63 ns, and fractional contributions of the four components were 2%, 16%, 52%, and 30%, respectively. This finding indicates that the environment of the tryptophan is modified by phosphorylation. In the absence of divalent metal ions, the observed three decay times of the CTnI complexed with cardiac troponin C (CTnC) remained unchanged, and addition of Ca2+ or Mg2+ resulted in only small changes in the lifetimes. When phosphorylated CTnI was complexed with CTnC, a large increase of the longest-lived component was observed: τ4 > 11 ns with its contribution shifted to 47%. Two rotational correlation times were observed for CTnI: φ1~0.9 ns and φ2~ 23.5 ns. These valves increased to t0~l.2 ns and ~30.1 ns, respectively, for the complex CtnI CTnC. Upon phosphorylation the two correlation times were significantly reduced regardless of whether CTnI was uncomplexed or complexed with CTnC. These results suggest that phosphorylation of CTnI resulted in a significantly more compact structure and enhanced motion of the tryptophan side chain. These structural changes may play a role in the transmission of Ca2+ signal in cardiac muscle. Thus, the effect of phosphorylation of CTnI becomes more pronounced when the protein is complexed with CTnC. These results suggest that there was likely a fluorophore heterogeneity which may arise from differences in conformation, environment, and/or different deactivation pathways for the excited state of the fluorophore.