Abstract: Titin functions as a molecular spring, and cardiomyocytes are able, through splicing, to control the length of titin. We hypothesized that together with diastolic [Ca2+], titin-based stretch pre-activates cardiomyocytes during diastole and is a major determinant of force production in the subsequent contraction. Through this mechanism titin would play an important role in active force development and length-dependent activation. Mutations in the splicing factor RNA binding motif protein 20 (RBM20) result in expression of large, highly compliant titin isoforms. We measured single cardiomyocyte work loops that mimic the cardiac cycle in wild-type (WT) and heterozygous (HET) RBM20-deficient rats. In addition, we studied the role of diastolic [Ca2+] in membrane-permeabilized WT and HET cardiomyocytes. Intact cardiomyocytes isolated from HET left ventricles were unable to produce normal levels of work (55% of WT) at low pacing frequencies, but this difference disappeared at high pacing frequencies. Length-dependent activation (force–sarcomere length relationship) was blunted in HET cardiomyocytes, but the force–end-diastolic force relationship was not different between HET and WT cardiomyocytes. To delineate the effects of diastolic [Ca2+] and titin pre-activation on force generation, measurements were performed in detergent-permeabilized cardiomyocytes. Cardiac twitches were simulated by transiently exposing permeabilized cardiomyocytes to 2 µm Ca2+. Increasing diastolic [Ca2+] from 1 to 80 nm increased force development twofold in WT. Higher diastolic [Ca2+] was needed in HET. These findings are consistent with our hypothesis that pre-activation increases active force development. Highly compliant titin allows cells to function at higher diastolic [Ca2+].