Lipopolysaccharide (LPS) has been implicated in sepsis-mediated heart failure and chronic cardiac myopathies. We determined that LPS directly and reversibly affects cardiac myocyte function by altering regulation of intracellular Ca2+ concentration ([Ca2+]i) in immortalized cardiomyocytes, HL-1 cells. [Ca2+]i oscillated (<0.4 Hz), displaying slow and transient components. LPS (1 μg/ml), derived either from Escherichia coli or from Salmonella enteritidis, reversibly abolished Ca2+ oscillations and decreased basal [Ca 2+]i by 30-40 nM. HL-1 cells expressed Toll-like receptors, i.e., TLR-2 and TLR-4. Thus, we differentiated effects of LPS on [Ca2+]i and Ca2+ oscillations by addition of utlrapure LPS, a TLR-4 ligand. Ultrapure LPS had no effect on basal [Ca 2+]i, but it reduced the rate of Ca2+ oscillations. Interestingly, Pam3CSK4, a TLR-2 ligand, affected neither Ca 2+ parameter, and the effect of ultrapure LPS and Pam3CSK4 combined was similar to that of utlrapure LPS alone. Thus, unpurified LPS directly inhibits HL-1 calcium metabolism via TLR-4 and non-TLR-4-dependent mechanisms. Since others have shown that endotoxin impairs the hyperpolarization-activated, nonselective cationic pacemaker current (If), which is expressed in HL-1 cells, we utilized whole cell voltage-clamp techniques to demonstrate that LPS (1 μg/ml) reduced If in HL-1 cells. This inhibition was marginal at physiologic membrane potentials and significant at very negative potentials (P < 0.05 at -140, -150, and -160 mV). So, we also evaluated effects of LPS on tail currents of fully activated If. LPS reduced the slope conductance of the tail currents from 498 ± 140 pS/pF to 223 ± 65 pS/pF (P < 0.05) without affecting reversal potential of -11 mV. Ultrapure LPS had similar effect on If, whereas Pam3CSK4 had no effect on If. We conclude that LPS inhibits activation of I f, enhances its deactivation, and impairs regulation of [Ca 2+]i in HL-1 cardiomyocytes via TLR-4 and other mechanisms.