It is well known that the stiffness of titin is the major determinant of the passive myocardial tension existing under relaxed conditions. To explore whether titin's intrinsic stiffness also determines the rate by which steady state relaxation is achieved, we determined the kinetics of force relaxation of cardiac myofibrils isolated from N2B-knockout mice (Radke et al., 2007). These mice express truncated cardiac titins which are stiffer than the full-length isoforms because of the deletion of titin's elastic N2B-domain. Left ventricular myofibrils from N2B-KO mice exhibit about 1.5-fold to 2-fold higher passive stiffness than myofibrils isolated from wildtype (WT). Relaxation kinetics were induced by rapidly reducing the [Ca²⁺] from pCa 4.5 to 7.5. Upon [Ca²⁺]-reduction, myofibrillar force decays biphasically, starting with a slow linear phase (rate constant k_LIN) lasting for a time (t_LIN) followed by a fast mono-exponential, relaxation phase (rate constant k_REL). Neither the kinetics of the slow nor of the fast relaxation phase were significantly affected by the deletion of the N2B-domains (k_LIN = 1.91 ± 0.13 s^-1 (n = 48), t_LIN = 0.046 ± 0.002 s (n = 48), k_REL = 34.14 ± 0.94 s^-1 (n = 48) for KO versus k_LIN = 2.06 ± 0.15 s^-1 (n = 47), t_LIN = 0.048 ± 0.002 s (n = 47), k_REL = 32.03 ± 1.04 s^-1 (n = 47) for WT). This suggests that increased titin-based stiffness does not slow down cardiac myofibrillar relaxation and implies that passive mechanical properties of the sarcomere do not directly influence relaxation kinetics.