The reactive SH1 (Cys-707) group of the myosin subfragment 1 (S1) has been used frequently as an attachment site for fluorescent and spin probes in solution and muscle fiber experiments. In this study we examined (i) the motor function of SH1 spin-labeled heavy meromyosin (HMM) in the in vitro motility assays and (ii) the effect of SH1-modified S1 on the motility of regulated actin, i.e., actin complexed with tropomyosin and troponin. N-ethylmaleimide (NEM), N-(1-oxyl-2,2,6,6-tetramethyl-4-piperidinyl)iodacetamide (IASL), N-[[(iodoacetyl)amino]ethyl]1-sulfo-5-naphthylamine (IAEDANS), and iodoacetamide (IAA) were used to selectively modify the SH1 group on S1; the SH1 group on HMM was labeled with IASL. In the in vitro motility assays, 10−20% of unregulated actin filaments moved at a speed of ∼1 μm/s over a surface coated with 90−95% modified IASL-HMM. Actin sliding was not observed with 95−98% modified IASL-HMM. The sliding of regulated actin over unmodified HMM was activated by the addition of S1 modified with any of the SH1 reagents to the in vitro motility assay solutions; both the speeds and the percentage of the moving filaments increased at pCa 5, 7, and 8. To shed light on the activation of regulated actin sliding by SH1-modifed S1, acto-S1 ATPase and the binding to actin were determined for IASL-S1. While the binding affinities to actin were similar for IASL-S1 and unmodified S1 in the presence and absence of ADP and ATP, the K_m and V_max values were approximately 10-fold lower for the modified protein. It is concluded that the activation of regulated actin by SH1-modifed S1 facilitates the interaction of unmodified HMM heads with actin and thus can increase the sliding speeds and the percentage of regulated actin filaments that move in the in vitro motility assays.