Vertebrate smooth muscle contraction is regulated by phosphorylation of the myosin regulatory light chains. EM studies of smooth muscle myosin molecules suggest that in the dephosphorylated state, activity is switched off by asymmetric interactions between the two myosin heads, in which the “blocked” head is thought to be unable to bind to actin, while the “free” head has its ATPase activity inhibited. Comparative sequence analysis and molecular modeling suggest that the head interaction may be stabilized by charge attraction between D748 in the converter domain of the free head and K368 of the blocked head, and between R406 or K416 of the blocked head and E169 of the free head (Jung et al., MBC, 2008). To test these possibilities, we investigated the structure of expressed smooth muscle myosin and heavy meromyosin in which the charges at some of these sites was neutralized or reversed. While most of the mutants showed no obvious change in structure compared with wild type, HMM with charge reversal at both 368 and 406 showed fewer molecules with interacting heads when observed by metal shadowing. This was supported by a decrease in sedimentation coefficient (determined by analytical ultracentrifugation) and an increase in actin-activated ATPase activity of the mutant. Whole myosin with the same mutations formed filaments more readily than wild type, also consistent with these results. These observations suggest that charge interactions involving K368 and R406 play a role in stabilizing the head-head interaction of the inactive state. Interestingly, mutation of R403 in cardiac myosin (R406 in smooth muscle) increases motor activity of myosin and causes familial hypertrophic cardiomyopathy. Our results suggest that this could be in part a result of changes in head interaction.