Recently we reported that Toll-like receptor 4 (TLR4)-positive immune cells of unknown identity were responsible for the LPS-induced depression of cardiac myocyte shortening. The aim of this study is to identify the TLR4-positive cell type that is responsible for the LPS-induced cardiac dysfunction. Neither neutrophil depletion alone nor mast cell deficiency had any impact on the impairment of myocyte shortening during LPS treatment. In contrast, LPS-treated, macrophage-deficient mice demonstrated a partial reduction in shortening compared with saline-treated, macrophage-deficient mice. Because the removal of macrophages could only partially restore myocyte shortening, we also investigated the effects of removing both neutrophils and macrophages on myocyte shortening. Interestingly, endotoxemic, neutrophil-depleted, and macrophage-deficient mice had completely restored myocyte shortening. Because both macrophages and neutrophils can produce nitric oxide (NO) and TNF-alpha, we examined LPS-treated inducible NO synthase knockout (iNOSKO) mice and TNF receptor (TNFR)-deficient mice. Eliminating both TNFR1 and TNFR2 was required to restore myocyte shortening during LPS treatment, whereas iNOS deficiency had no effect. These data suggest that macrophages and to a lesser degree neutrophils cause cardiac impairment, presumably via TNF-alpha.