Myosinopathies have emerged as a new group of diseases caused by mutations in genes encoding myosin heavy chain (MyHC) isoforms. One major hallmark of these diseases is skeletal muscle weakness or paralysis, but the underlying molecular mechanisms remain unclear. Here, we have undertaken a detailed functional study of muscle fibers from Myh4(arl) mice, which carry a mutation that provokes a L342Q change within the catalytic domain of the type IIb skeletal muscle myosin protein MYH4. Since the rapid muscle structure disruption and lower limb paralysis that homozygous animals develop demands that they are killed by postnatal day 13, all experiments were performed using skeletal muscles from adult heterozygous animals (Myh4(arl)/+). Myh4(arl)/+ mice contain MYH4(L342Q) expressed at 7% of the levels of the wild-type protein and are overtly and histologically normal. However, mechanical and X-ray diffraction pattern analyses of single membrane-permeabilized fibers revealed, upon maximal Ca(2+) activation, higher stiffness as well as altered meridional and equatorial reflections in Myh4(arl)/+ when compared with age-matched wild-type animals (WT). Under rigor conditions, on the other hand, no difference was observed between Myh4(arl)/+ and WT. Altogether, these findings prove that in adult heterozygous mice, in the presence of the MYH4(L342Q) mutation, myosin cross-bridge weak to strong binding transition is facilitated, increasing the number of strongly attached myosin heads, thus enhancing force production. These changes are predictably exacerbated in the type IIb fibers of homozygous mice in which the embryonic myosin isoform is fully replaced by MYH4(L342Q), leading to a hyper contraction, muscle structure disruption and lower limb paralysis. Overall, these findings provide important insights into the molecular pathogenesis of skeletal myosinopathies.