Heterotrimeric G(i) proteins may play a role in lipopolysaccharide (LPS)-activated signaling through Toll-like receptor 4 (TLR4), leading to inflammatory mediator production. Although LPS is a TLR4 ligand, the gram-positive bacterium Staphylococcus aureus (SA) is a TLR2 ligand, and group B streptococci (GBS) are neither TLR2 nor TLR4 ligands but are MyD88 dependent. We hypothesized that genetic deletion of G(i) proteins would alter mediator production induced by LPS and gram-positive bacterial stimulation. We examined genetic deletion of Galpha(i2) or Galpha(i1/3) protein in Galpha(i2)-knockout (Galpha(i2)-/-) or Galpha(i1/3)-knockout (Galpha(i1/3)-/-) mice. LPS-, heat-killed SA-, or GBS-induced mediator production in splenocytes or peritoneal macrophages (MPhi) was investigated. There were significant increases in LPS-, SA-, and GBS-induced production of TNF-alpha and IFN-gamma in splenocytes from Galpha(i2)-/- mice compared with wild-type (WT) mice. Also, LPS-induced TNF-alpha was increased in splenocytes from Galpha(i1/3)-/- mice. In contrast to splenocytes, LPS-, SA-, and GBS-induced TNF-alpha, IL-10, and thromboxane B(2) (TxB(2)) production was decreased in MPhi harvested from Galpha(i2)-/- mice. Also, LPS-induced production of IL-10 and TxB(2) was decreased in MPhi from Galpha(i1/3)-/- mice. In subsequent in vivo studies, TNF-alpha levels after LPS challenge were significantly greater in Galpha(i2)-/- mice than in WT mice. Also, myeloperoxidase activity, a marker of tissue neutrophil infiltration, was significantly increased in the gut and lung of LPS-treated Galpha(i2)-/- mice compared with WT mice. These data suggest that G(i) proteins differentially regulate murine TLR-mediated inflammatory cytokine production in a cell-specific manner in response to both LPS and gram-positive microbial stimuli.