Familial hypertrophic cardiomyopathy is a disease characterized by left ventricular and/or septal hypertrophy and myofibrillar disarray. It is caused by mutations in the sarcomeric proteins including the ventricular isoform of myosin regulatory light chain (RLC). The E22K mutation is located in the RLC Ca²⁺ binding site. In this work, we studied transgenic mouse cardiac myofibrils during single-turnover contraction, to examine the influence of E22K mutation on: (i) dissociation time, ₁, of myosin heads from thin filaments, (ii) re-binding time, ₂, of the cross-bridges to actin, and (iii) the dissociation time, ₃, of ADP from the active site of myosin. Cross-bridges were induced to undergo a single detachment-attachment cycle by a precise delivery of stoichiometric ATP from a caged precursor. The times were measured in transgenic-mutated (Tg-m) heart myofibrils overexpressing the E22K mutation of human cardiac RLC. Transgenic wild type (Tg-wt) and non-transgenic (non-Tg) muscles acted as controls. ₁ was statistically greater in Tg-m than in controls. ₂ was shorter in Tg-m than in non-Tg, but the same as in Tg-wt. ₃ was the same in Tg-m and in controls. In an effort to see if the observed difference in ₁ was due to intrinsic difference in myosin, binding of Tg-m and Tg-wt myosin to fluorescently labeled actin was estimated by measuring fluorescent lifetime and time-resolved anisotropy. No difference in binding was observed. These results suggest that the E22K mutation has no effect on mechanical properties of cross-bridges. Slight increase in ₁ was probably caused by myofibrillar disarray.