In spite of extensive work on the functional sequelae of HCM-associated mutations in sarcomeric proteins, the mechanisms by which the mutant proteins cause the disease have not been definitely identified. Here we use the single myofibril technique (Tesi et al., Biophys. J., 2002, 83, 2142-2151) to compare the kinetics of contraction and relaxation of myofibrils isolated from frozen left ventricular samples of one homozygous HCM patient carrying the K280N cTnT mutation (underwent heart transplantation) to those from "control" hearts. Preparations, mounted in a force recording apparatus (15 °C), were maximally Ca2+-activated (pCa 4.5) and fully relaxed (pCa 9) by rapid (< 10 ms) solution switching. The rate constant of active tension generation following maximal Ca2+ activation (kACT) was markedly faster in the myofibrils from the K280N patient (1.7- 2 s-1) compared to controls (0.7-1 s−1). Force relaxation kinetics upon Ca2+ removal were also faster in K280N myofibrils; the rate constant of isometric relaxation (slow kREL) was 2-3 times faster in K280N myofibrils than in controls, evidence that the apparent rate with which cross-bridges leave the force generating states is accelerated in the HCM preparations. Replacement of the mutant protein by exchange with wild-type recombinant human Tn reduced both kACT and slow kREL of HCM myofibrils close to control values. The results indicate that the HCM-associated cTnT mutation K280N alters apparent cross-bridge kinetics. This can lead to increased energy cost of tension generation that may be central to the HCM disease process. Supported by the 7th Framework Program of the European Union, "BIG-HEART" grant agreement 241577, & Telethon-Italy GGP07133.