We have recently identified a new state of myosin in relaxed muscle, the “super-relaxed state” (SRX), with a very slow ATP turnover rate. The SRX has now been found in five muscles: fast and slow skeletal muscle, cardiac muscle, smooth muscle and tarantula leg muscle, suggesting it is a fundamental characteristic of the relaxed state. In all fiber types the SRX provides a state that has a more economical ATPase activity than the relaxed state in which myosin heads are disordered. It has been proposed that myosin heads in the SRX are inhibited by binding to the core of the thick filament in a structure known as the “interacting heads motif” (IHM). In the IHM the two regulatory light chains (RLCs) bind to each other. To understand this interface, we have made single cysteine mutants of the RLC, placed both paramagnetic and fluorescent probes on them, and exchanged them into skeletal muscle fibers. Probes were located within the putative RLC-RLC interface tended to disrupt the stability of the SRX. Probes far from this interface had no effect on the SRX. Most of the spin-labels bound to the RLC were disordered in all states, rigor, disordered relaxed state and SRX, however one label showed good orientation in the SRX. Some fluorescent probes adjacent to the putative interface showed spectral shifts between the disordered relaxed state and the SRX. Together the results provide further evidence identifying the location of the RLC-RLC interface, and provide a tool for further structural studies.