Fluorescently labeled myosin heads (S1) were added to muscle fibers and myofibrils at various concentrations. The orientation of the absorption dipole of the dye with respect to the axis of F-actin was calculated from polarization of fluorescence which was measured by a novel method from video images of muscle. In this method light emitted from muscle was split by a birefringent crystal into two nonoverlapping images: the first image was created with light polarized in the direction parallel to muscle axis, and the second image was created with light polarized in the direction perpendicular to muscle axis. Images were recorded by high-sensitivity video camera and polarization was calculated from the relative intensity of both images. The method allows measurement of the fluorescence polarization from single myofibril irrigated with low concentrations of S1 labeled with dye. Orientation was also measured by fluorescence-detected linear dichroism. The orientation was different when muscle was irrigated with high concentration of S1 (molar ratio S1:actin in the I bands equal to 1) then when it was irrigated with low concentration of S1 (molar ratio S1:actin in the I bands equal to 0.32). The results support our earlier proposal that S1 could form two different rigor complexes with F-actin depending on the molar ratio of S1:actin.