Transgenic (Tg) mice expressing approximately 95% of the D166V (aspartic acid to valine) mutation in the ventricular myosin regulatory light chain (RLC) shown to cause a malignant familial hypertrophic cardiomyopathy (FHC) phenotype were generated, and the skinned and intact papillary muscle fibers from the Tg-D166V mice were examined using a Guth muscle research system. A large increase in the Ca(2+) sensitivity of force and ATPase (Delta pCa(50)>0.25) and a significant decrease in maximal force and ATPase were observed in skinned muscle fibers from Tg-D166V mice compared with control mice. The cross-bridge dissociation rate g was dramatically decreased, whereas the energy cost (ATPase/force) was slightly increased in Tg-D166V fibers compared with controls. The calculated average force per D166V cross-bridge was also reduced. Intact papillary muscle data demonstrated prolonged force transients with no change in calcium transients in Tg-D166V fibers compared with control fibers. Histopathological examination revealed fibrotic lesions in the hearts of the older D166V mice. Our results suggest that a charge effect of the D166V mutation and/or a mutation-dependent decrease in RLC phosphorylation could initiate the slower kinetics of the D166V cross-bridges and ultimately affect the regulation of cardiac muscle contraction. Profound cellular changes observed in Tg-D166V myocardium when placed in vivo could trigger a series of pathological responses and result in poor prognosis for D166V-positive patients.