Skeletal muscle fibers produce nitric oxide (^·NO) and myosin has a large number of free thiol groups that can be S-nitrosylated by GSNO, affecting its catalytic properties (Nogueira et al, SBBq 2003). Here we investigate whether –SNO bonds on myosin have different reactivities and differ in their ability to affect Mg²⁺ATPase and actin-activated activity. Myosin (2mg/ml) was treated with GSNO (0-8mM) for 15min in Mg²⁺ buffer (50mM KCl, 50mM imidazol pH7, 0.2mM EDTA, 5mM MgCl₂; 30ºC) followed by dilution (1:3.5 v/v) with cold water and centrifugation to remove excess GSNO and reaction products (1-5 cycles of separation). Formation of –SNO bonds was verified using Griess reagent + HgCl₂ and myosin activity was measured by colorimetric determination of P_i. After the reaction with 3mM GSNO, there was a progressive decrease in –SNO bonds from 25 to 10 to 2.5 mol/mol in response to 1, 2 and 5 cycles of separation and the inhibitory effect on Mg²⁺ATPase activity was diminished in parallel. There were no differences in inhibition between 1 cycle of separation and the absence of GSNO separation, suggesting that the more labile –SNO bonds are responsible for the inhibition. To test whether the more stable –SNO groups are located in peripheral regions or in the core of the protein, myosin was treated with 1mM NEM followed by 10mM DTT to block the more acessible thiols in the protein. After extensive dialysis, NEM-myosin was treated with DTNB (pH7, 25ºC, 2h) to determine the number of accessible thiols, which was reduced to the same extent with and without GndCl. In separate experiments, NEM-myosin reacting with low [GSNO] followed by 5 cycles of separation, formed the same number of –SNO bonds (0.5-2.5 mol –SNO/mol myosin) as the native protein, but half of the –SNO bonds formed were blocked at high [GSNO] (>3mM), suggesting that the more stable –SNO bonds (2.5mol/mol) are in the core of the protein, which is less accessible to NEM. To verify GSNO effects on actin-activated activity, myosin was treated with GSNO followed by 2 cycles of separation and added to actin medium with ATP. There was no effect of GSNO on acto-myosin affinity, but the maximal actin-activation was reduced »20%. We infer that ^·NO can regulate myosin and actomyosin activity transiently by S-nitrosylation forming labile –SNO bonds, perhaps when NOS activity is increased during muscle contraction or exercise.