—A mutation in the cardiac β-myosin heavy chain, Arg403Gln (R403Q), causes a severe form of familial hypertrophic cardiomyopathy (FHC) in humans. We used small-amplitude (0.25%) length-perturbation analysis to examine the mechanical properties of skinned left ventricular papillary muscle strips from mouse hearts bearing the R403Q mutation in the α-myosin heavy chain (αMHC^403/+). Myofibrillar disarray with variable penetrance occurred in the left ventricular free wall of the αMHC^403/+ hearts. In resting strips (pCa 8), dynamic stiffness was ≈40% greater than in wild-type strips, consistent with elevated diastolic stiffness reported for murine hearts with FHC. At pCa 6 (submaximal activation), strip isometric tension was ≈3 times higher than for wild-type strips, whereas at pCa 5 (maximal activation), tension was marginally lower. At submaximal calcium activation the characteristic frequencies of the work-producing (b) and work-absorbing (c) steps of the crossbridge were less in αMHC^403/+ strips than in wild-type strips (b=11±1 versus 15±1 Hz; c= 58±3 versus 66±3 Hz; 27°C). At maximal calcium activation, strip oscillatory power was reduced (0.53±0.25 versus 1.03±0.18 mW/mm³; 27°C), which is partly attributable to the reduced frequency b, at which crossbridge work is maximum. The results are consistent with the hypothesis that the R403Q mutation reduces the strong binding affinity of myosin for actin. Myosin heads may accumulate in a preforce state that promotes cooperative activation of the thin filament at submaximal calcium but blunts maximal tension and oscillatory power output at maximal calcium. The calcium-dependent effect of the mutation (whether facilitating or debilitating), together with a variable degree of fibrosis and myofibrillar disorder, may contribute to the diversity of clinical symptoms observed in murine FHC.