Disease-causing mutations in cardiac myosin heavy chain (beta-MHC) are identified in about one-third of families with hypertrophic cardiomyopathy (HCM). The effect of myosin mutations on calcium sensitivity of the myofilaments, however, is largely unknown. Because normal and mutant cardiac MHC are also expressed in slow-twitch skeletal muscle, which is more easily accessible and less subject to the adaptive responses seen in myocardium, we compared the calcium sensitivity (pCa(50)) and the steepness of force-pCa relations (cooperativity) of single soleus muscle fibers from healthy individuals and from HCM patients of three families with selected myosin mutations. Fibers with the Arg723Gly and Arg719Trp mutations showed a decrease in mean pCa(50), whereas those with the Ile736Thr mutation showed slightly increased mean pCa(50) with higher active forces at low calcium concentrations and residual active force even under relaxing conditions. In addition, there was a marked variability in pCa(50) between individual fibers carrying the same mutation ranging from an almost normal response to highly significant differences that were not observed in controls. While changes in mean pCa(50) may suggest specific pharmacological treatment (e.g., calcium antagonists), the observed large functional variability among individual muscle cells might negate such selective treatment. More importantly, the variability in pCa(50) from fiber to fiber is likely to cause imbalances in force generation and be the primary cause for contractile dysfunction and development of disarray in the myocardium.