SH-1 thiol of S-1 was modified with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) iodoacetoamide spin label (IASL). The extent of dissociation, alpha, of spin-labeled myosin subfragment-1 (IASL-S-1) from acto-IASL-S-1 by a nucleotide was measured by an ultracentrifugal separation method, a light-scattering method, and a saturation transfer EPR method. The alpha values obtained by these three methods were the same within the limits of the experimental errors. The dependence of alpha on the concentrations of AMPPNP, [S], and F-actin, [A], could be described by the equation: alpha-1 = 1 + (1 + Ks/[S])[A]/KA. The Ks and KA values were 0.65-1.2 mM and 1.7-2.7 mg/ml, respectively, in 0.5 M KCl and 4 mM MgCl2 at pH 7.0 and 20 degrees C. The height of the weakly immobilized peak of the conventional EPR spectrum of IASL-S-1, W, increased linearly with increase in the ATP or AMPPNP concentration, and became saturated at 1 mol nucleotide/mol IASL-S-1. No change in W was observed upon the binding of IASL-S-1 with F-actin. The dependence of the extent of change in W, delta W, on [A] and [S] was given by delta W-1 = 1 + Ks/[S], where Ks = Ks/(1 + KA/[A]). This finding indicates that the delta W value is proportional to the amount of a nucleotide bound to IASL-S-1 and independent of the binding of F-actin to IASL-S-1.