Two cardiomyopathic mutations were expressed in human cardiac actin, using a Baculovirus/insect cell system; E99K is associated with hypertrophic cardiomyopathy whereas R312H is associated with dilated cardiomyopathy. The hypothesis that the divergent phenotypes of these two cardiomyopathies is associated with fundamental differences in the molecular mechanics and thin filament regulation of the underlying actin mutation was tested using the in vitro motility and laser trap assays. In the presence of troponin (Tn) and tropomyosin (Tm), β-cardiac myosin moved both E99K and R312H thin filaments at significantly (p<0.05) slower velocities than wild type (WT) at maximal Ca++. At submaximal Ca++, R312H thin filaments demonstrated significantly increased Ca++ sensitivity (pCa50) when compared to WT. Velocity as a function of ATP concentration revealed similar ATP binding rates but slowed ADP release rates for the 2 actin mutants compared to WT. Single molecule laser trap experiments performed using both unregulated (i.e. actin) and regulated thin filaments in the absence of Ca++ revealed that neither actin mutation significantly affected the myosin’s unitary step size (d) or duration of strong actin binding (ton) at 20 μM ATP. However, the frequency of individual strong-binding events in the presence of Tn and Tm, was significantly lower for E99K than WT at comparable myosin surface concentrations. The cooperativity of a second myosin head binding to the thin filament was also impaired by E99K. In conclusion, E99K inhibits the activation of the thin filament by myosin strong-binding whereas R312H demonstrates enhanced calcium activation.