Recently we showed that the Familial Hypertrophic Cardiomyopathy associated mutation R145G in cardiac troponin I (cTnI) slows down the kinetics of force relaxation in cardiac myofibrils (cMF). To determine whether this was due to reduced switch-off kinetics of Tn we developed a new technique to measure switch-off kinetics of Tn in cMF, i.e. within the structural constrains imposed by the sarcomere. Site directed mutagenesis of C35S in cTnC allowed us to label specifically the remaining C84 in cTnC with the fluorescence dye IANBD. The labelled cTnC was reconstituted with cTnI and cTnT to the cTn complex and incorporated in cMF. cMF were Ca²⁺-activated in a stopped-flow apparatus for 100 ms and then mixed with excess BAPTA to rapidly lower (< 1ms) Ca²⁺ and, thereby, switch off Tn in cMF. Fluorescence decreased monoexponentially with a rate constant of 33.0 ± 3.1s^-1 (n=11) after Ca²⁺-removal. Preliminary results suggest that the mutation cTnI^R145G reduces the off-kinetics to 20.5 ± 1.1s^-1 (n=3) which may account for the decrease in the rate of force relaxation.