A GFP label was attached to the C-terminus end of wild type-human cardiac regulatory light chain HCRLC. The resulting HCRLC-GFP was exchanged into single rabbit skeletal muscle fibers. Total internal reflection fluorescence (TIRF) microscopy and image analysis signaled proper orientation of the probe in the myosin. Initial isometric contraction studies show normal force activation from skeletal muscle fibers exchanged with HCRLC-GFP. The GFP probe on (HCRLC) can be used to develop a better understanding of the function of HCRLC and other regulatory light chain (RLC) isoforms. Furthermore, the use of GFP-labeled HCRLC may be applied in vivo in model organisms such as zebra fish to further study both native and mutated HCRLC. This becomes particularly useful in cardiac disease studies such as hypertrophic cardiomyopathy, which has been associated with certain mutations in HCRLC.