We have labeled rabbit skeletal muscle actin with the triplet probe erythrosin-5-iodoacetamide and characterized the labeled protein. Labeling decreased the critical concentration and lowered the intrinsic viscosity of F-actin filaments; labeled filaments were motile in an in vitro motility assay but were less effective than unlabeled F-actin in activating myosin S1 ATPase activity. In unpolymerized globular actin (G-actin), both the prompt and delayed luminescence were red-shifted from the spectra of the free dye in solution and the fluorescence anisotropy of the label was high (0.356); filament formation red shifted all excitation and emission spectra and increased the fluorescence anisotropy to 0.370. The erythrosin phosphorescence decay was at least biexponential in G-actin with an average lifetime of 99 microseconds while in F-actin the decay was approximately monoexponential with a lifetime of 278 microseconds. These results suggest that the erythrosin dye was bound at the interface between two actin monomers along the two-start helix. The steady-state phosphorescence anisotropy of F-actin was 0.087 at 20 degrees C and the anisotropy increased to approximately 0.16 in immobilized filaments. The phosphorescence anisotropy was also sensitive to binding the physiological ligands phalloidin, cytochalasin B and tropomyosin. This study lays a firm foundation for the use of this triplet probe to study the large-scale molecular dynamics of F-actin.