The fluorophore, N(iodoacetylamino)-1-naphthylamine-5-sulfonic acid (1,5-IAEDANS), incubated with glycerinated psoas fibers primarily labels the S-1 moieties of such fibers, but it does not impair fiber contractility even when the degree of labeling is as high as 0.8 moles fluorophore per mole myosin. The polarization of the on-axis fluorescence from either the IAEDANS fluorophore, or the intrinsic tryptophane fluorophore, depends on whether the fiber is relaxed, in rigor, or developing isometric tension; furthermore, the changes in polarization on going from one state to another are much the same with either tryptophane or IAEDANS fluorophores. The foregoing is true whether the plane of the exciting light is parallel or perpendicular to the fiber axis. Also, if a fiber is first freed of its myosin by extraction, and is then incubated with IAEDANS-labeled S-1 the resulting polarization approaches that observed with a labeled, unextracted fiber in rigor. By contrast, incubation with the fluorophore, 7-nitro-4-chlorobenz-2-oxa-1,3-diazole (NBD-Cl) confers fluorescence only on actin, without impairing contractility, but the polarization of such fluorescence changes in a different direction and magnitude from myosin-originating fluorescence. It is concluded from these various observations that whether the fluorophore is IAEDANS or tryptophane the polarization change with change in physiological state originates in the S-1 moieties of fibers, and relates to the space attitude of these moieties.