N-ethylmaleimide (NEM) treatment of myosin inhibits relaxation in actomyosin systems from rabbit skeletal muscle which appear to be regulated solely through tropomyosin [Tm] and troponin [T]. Rigor complexes between (nucleotide-free) myosin and actin affect the T-Tm system. As a result of NEM treatment, some of the myosin maintains rigor complexes with actin in the presence of ATP and are responsible for inhibition of relaxation. After exhaustive NEM treatment of heavy meromyosin (HMM), acto-HMM complexes were no longer dissociated by ATP. Admixture of such NEM-treated, enzymatically inactive HMM or myosin, to native regulated actomyosin or acto-HMM, inhibited relaxation.