The orientation of the regulatory light chain (RLC) region of the myosin heads in relaxed skinned fibers from rabbit psoas muscle was investigated by polarized fluorescence. Mutants of chicken skeletal RLC were labeled by crosslinking cysteine pairs with bifunctional sulphorhodamine (BSR). BSR-RLCs were exchanged into skinned muscle fibers replacing about 15% of the native RLC. The second- and fourth-rank order parameters of the orientation distribution of each BSR-RLC in the fiber, and respectively, were calculated from the measured polarized fluorescence intensities. and had an approximately sigmoidal dependence on temperature in the range 5°C to 30°C in relaxed fibers at sarcomere length 2.5 mm, similar to that of the myosin-based layer lines observed by X-ray diffraction, and indicated that the RLC regions of the myosin heads became much more ordered at higher temperature. The ordered state observed at the higher temperature was reversibly abolished by stretching the relaxed fibers to sarcomere length 4.0 mm. These results suggest that the native helically-ordered distribution of the myosin heads on the surface of the thick filaments in relaxed mammalian muscle has been preserved in the presence of the BSR-RLC probes, opening the possibility that the data from probe studies of this type can be used to construct detailed structural models of the conformation of the RLC region of the myosin heads in situ. Supported by MRC and Wellcome Trust, UK.