Relaxing-factor activity of guinea-pig gastrocnemius muscle, as determined by the amount of calcium binding to microsomes, was found to rise significantly after denervation. This increase began 2-4 days following sciatic nerve section and reached a maximum on the 15th to the 17th day, when the amount of calcium binding was three times the value of the opposite, nondenervated gastrocnemius. This finding suggests that the biochemical mechanism for coupling excitation with contraction and relaxation is facilitated in denervated skeletal muscle and that such facilitation may play a role in producing fibrillations.