F-actin in a glycerinated muscle fiber was specifically labeled with fluorescent phalloidin-(fluorescein isothiocyanate) FITC complex at 1:1 molar ratio. Binding of phalloidin-FITC to F-actin affected neither contraction of the fiber nor its regulation by Ca2+. Comparison of polarized fluorescence from phalloidin-FITC bound to F-actin in the relaxed state, rigor, and during isometric contraction of the fiber revealed that the changes in polarization accompanying activation are quantitatively as well as qualitatively different from those accompanying transition of the fiber from the relaxed state to rigor. The extent of the changes of polarized fluorescence during isometric contraction increased with decreasing ionic strength, in parallel with increase in isometric tension. On the other hand, polarized fluorescence was not affected by addition of ADP or by stretching of the fiber in rigor solution. It is concluded from these observations that conformational changes in F-actin are involved in the process of active tension development.