Lipopolysaccharide (LPS) binds to Toll-like receptor 4 (TLR4) on immune cells but also on cardiac myocytes and leads to sepsis with all accompanied effects such as cardiac depression, decreasing blood pressure and finally septic shock. It is discussed whether cardiac depression is caused by a decreased Ca²⁺transient or a reduced Ca²⁺-sensitivity of the myofilaments. To clarify this we isolated cardiac myocytes of C57BL/6 (WT) and TLR4-deficient (TLR4^-/-) mice and recorded sarcomere shortening and intracellular Ca²⁺-concentration ([Ca²⁺]_i) simultaneously. [Ca²⁺]_i was recorded by means of fura-2 fluorescence. Cells were stimulated externally at 4 Hz after incubation in short-term culture in the presence or absence of 1 mg/ml LPS for > 5 h. To detect LPS dependent pathways and its influence on [Ca²⁺]_i S-Methylthiourea (SMT), an iNOS inhibitor, was added to WT cells. Sarcomere shortening was not significantly changed after 5 h of culture but the amplitude of [Ca²⁺]_i was significantly decreased. Stimulation with LPS led to a significant suppression of sarcomere shortening amplitude but did not affect [Ca²⁺]_i compared to cultured control myocytes. Addition of SMT prevented LPS dependent depression of sarcomere shortening but did not influence [Ca²⁺]_i compared to cultured control cells. In case of TLR4^-/-myocytes LPS did neither change sarcomere shortening nor [Ca²⁺]_i. Normalizing sarcomere shortening to [Ca²⁺]_i revealed that LPS treated myocytes were less sensitive to [Ca²⁺]_i.