Skeletal muscles present a non-crossbridge increase in sarcomere stiffness and tension upon Ca²⁺ activation, referred to as static stiffness and static tension, respectively. It has been hypothesized that this increase in tension is caused by Ca²⁺-dependent changes in the properties of titin molecules. To verify this hypothesis, we investigated the static tension in muscles containing different titin isoforms. Permeabilized myofibrils were isolated from the psoas, soleus and heart ventricle from the rabbit, and tested in pCa 9.0 and pCa 4.5, before and after extraction of troponin C, thin filaments and treatment with the acto-myosin inhibitor blebbistatin. The myofibrils were tested with stretches of different amplitudes in sarcomere lengths (SLs) varying between 1.93-3.37µm for the psoas, 2.68-4.21µm for the soleus, and 1.51µm-2.86µm for the ventricle. Using gel electrophoresis, we confirmed that the three muscles tested have different titin isoforms. The static tension was present in psoas and soleus myofibrils, but not in ventricle myofibrils, and higher in psoas myofibrils than in soleus myofibrils. These results suggest that the increase in the static tension is directly associated with Ca²⁺-dependent change in titin properties, and not associated with changes in titin-actin interactions.