Small (N2B) and large (N2BA) cardiac titin isoforms are differentially expressed in a species-specific and heart location-specific manner. To understand how differential expression of titin isoforms may influence passive stiffness of cardiac muscle we investigated the mechanical properties of mouse left ventricular (MLV) wall muscle (expressing predominantly the small titin isoform), bovine left atrial (BLA) wall muscle (predominantly the large isoform), and bovine left ventricular (BLV) wall muscle (expressing small and large isoforms at similar levels). Results indicate that the overall passive muscle stiffness of the muscle types varies nearly ten-fold, with stiffness increasing in the following order: BLA, BLV and MLV. To investigate the basis of the variation in the overall muscle stiffness, the contributions of titin and collagen to muscle stiffness were determined. Results showed that increased muscle stiffness results from increases in both titin- and collagen-based passive stiffness, indicating that titin and collagen change in a co-ordinated fashion. The expression level of the small titin isoform correlates with titin's contribution to overall muscle stiffness, suggesting that differential expression of titin isoforms is an effective means to modulate the filling behavior of the heart.