The aim of this study was to test if titin is a calcium-dependent spring and if it is the source of the passive force enhancement observed in muscle and single fibre preparations. We measured passive force enhancement in troponin C (TnC) depleted myofibrils in which active force production was completely eliminated. The TnC depleted construct allowed for investigation of the effect of calcium concentration on passive force, without the confounding effects of actin-myosin cross-bridge formation and active force production. Passive forces in TnC-depleted myofibrils (n=6) were 35.0 ± 2.9 nN/µm² when stretched to an average sarcomere length of 3.4 μm in a solution with low calcium concentration (pCa=8.0). Passive forces in the same myofibrils increased by 25 to 30% when stretches were performed in a solution with high calcium concentration (pCa=3.5). Since it is well accepted that titin is the primary source for passive force in rabbit psoas myofibrils, and since the increase in passive force in TnC-depleted myofibrils was abolished after trypsin treatment, our results suggest that increasing calcium concentration is associated with increased titin stiffness. However, this calcium-induced titin stiffness accounted for only about 25% of the passive force enhancement observed in intact myofibrils. Therefore, approximately 75% of the normally occurring passive force enhancement remains unexplained. The findings of this study suggest that passive force enhancement is partly caused by a calcium-induced increase in titin stiffness, but also requires cross bridge formation and/or active force production for full manifestation.