Exposure of macrophages to endotoxin (LPS) results in a cascade of events resulting in the release of multiple inflammatory and anti-inflammatory mediators. The Toll-like receptor (TLR) 4 complex is the major receptor that mediates LPS signaling. However, there is evidence that other surface molecules may play a complementary role in the TLR-induced events. Integrin receptors are one class of receptors that have been linked to LPS signaling. This study investigates the role of macrophage integrin receptors in the activation of MAP kinases by LPS. We found that in conditions where macrophages were not permitted to adhere to matrix or a tissue culture surface there was a decrease in LPS signaling as documented by a marked reduction in tyrosine phosphorylation of whole cell proteins. This was accompanied by a significant decrease in ERK and JNK MAP kinase activation. Inhibition of integrin signaling, with EDTA or RGD peptides, decreased LPS-induced MAP kinase activity. The functional consequence of blocking integrin signaling was demonstrated by decreased LPS-induced TNF- production. These observations demonstrate that in addition to the TLR receptor complex, optimal LPS signaling requires complementary signals from integrin receptors.