This study sought to determine whether gentamicin, a mainstay in treating Gram negative sepsis, alters endotoxin (lipopolysaccharide, LPS)- driven TNF-increases. Methods: CD-1 mice received 1 day of gentamicin treatment. Either 0, 24, or 72 hrs later, gentamicin treated- and control mice were injected with LPS. Renal cortical and plasma TNF-, as well as MCP-1, protein levels were measured 2 hrs or 24 hrs post LPS injection. Renal cortical mRNAs for TNF-, MCP-1, IL-10, and iNOS were also determined. Finally, potential gentamicin effects on TNF- / MCP-1 mRNA levels in non traditional target organs (liver, spleen) were assessed. Results: Gentamicin, when administered alone, slightly increased renal cortical TNF- and MCP-1 mRNAs, but without changing plasma or renal TNF- / MCP-1 protein levels. The gentamicin protocol induced no overt renal damage (assessed by BUNs, creatinines, and histology). Nevertheless, gentamicin augmented LPS responsiveness, as manifested, in part, by a doubling of LPS- induced plasma TNF- increases (vs. LPS injection alone). Plasma and renal cortical MCP-1 protein levels were also selectively enhanced. Gentamicin augmented LPS- driven renal mRNA increases (TNF-, MCP-1, IL-10, iNOS). However, this was not an entirely renal- specific response, since gentamicin also enhanced basal- and LPS- stimulated hepatic TNF- mRNA levels. Conclusions: Subclinical gentamicin toxicity can potentiate LPS- driven TNF- increases. Alterations in multiple pro- (TNF- MCP-1; iNOS) and anti- (IL-10) inflammatory genes in kidney, and possibly in extra-renal organs, may be involved. Thus, the impact of gentamicin on Gram negative sepsis may extend beyond its traditional antimicrobial effect.