Myosin has been spin labeled at the S 2 thiol groups, whose modification leads to loss of Ca 2+-ATPase activity, with N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl) maleimide after blocking of more rapidly reacting groups with NEM or DTNB. A loss of K +-ATPase activity and an increase in Ca 2+-ATPase accompany the reaction with NEM or DTNB; subsequent spin labeling produces a loss of Ca 2+-ATPase activity which is linearly related to the amount of spin label bound and is complete with 2.1 moles of label bound per 500,000 g of myosin. After sequential reaction with DTNB and the spin label, Ca 2+-ATPase activity can be restored by treatment with DTT with no change in the ESR spectrum. The S 2 groups differ from S 1 groups in their reactivity; an iodoacetamide spin label which reacts rapidly and preferentially with S 1 groups of native myosin does not react readily with S 2 groups of NEM- or DTNB-blocked myosins. In addition, the rate of reaction of S 1 groups with the iodoacetamide label is unaffected by ADP while the reaction of S 2 groups with the maleimide label is greatly accelerated by ADP. Spin labels bound to S 2 groups are strongly immobilized. Their mobility is increased by ATP, ITP, ADP, or PP i, but this effect is somewhat smaller than that previously observed with myosin labeled at the S 1 groups. The change in the ESR spectrum of S 2 labeled myosin induced by ATP reflects binding of the nucleotide since it does not require hydrolysis and the same effect is produced by ADP. The additional spectral change observed during ATP hydrolysis with S 1-labeled myosin is not observed with S 2-labeled myosin. If S 2 groups are labeled with a series of maleimide labels in which the number of atoms between the maleimide and nitroxide rings differ, increasing the distance between the two rings of the spin label results in a transition from strong immobilization to weak immobilization. Adenosine triphosphate appears to affect the mobility of only those labels which are strongly immobilized.