Gentamicin is a mainstay in treating Gram negative sepsis. However, it may also potentiate endotoxin (LPS) driven- plasma TNF- increases. Because gentamicin accumulates in renal tubules, this study addressed whether gentamicin directly alters LPS- driven tubular cell TNF- production. Methods: HK-2 proximal tubular cells were incubated for 18 hr with gentamicin (10-2000 µmicrogm/ml). Subsequent LPS- mediated TNF- increases (at 3 or 24 hrs; protein / mRNA) were determined. Gentamicin effects on overall protein synthesis (³⁵S methionine incorporation), MCP-1 levels, and LPS stimulated- TNF- generation by isolated mouse proximal tubules also were assessed. Finally, because gentamicin undergoes partial biliary excretion, its potential influence on gut TNF-/MCP-1 mRNAs was probed. Results: Gentamicin caused striking, dose dependent, inhibition of LPS- driven TNF- production (up to 80% in HK-2 cells / isolated tubules). Surprisingly, this occurred despite increased TNF- mRNA accumulation. Comparable changes in MCP-1 were observed. These were expressed at clinically relevant gentamicin concentrations, and despite essentially normal overall protein synthetic rates. Streptomycin also suppressed LPS- driven TNF- increases, suggesting an aminoglycoside drug class effect. Gentamicin doubled basal TNF- mRNA in cecum, and in small intestine after LPS. Conclusions: Gentamicin can suppress LPS- driven TNF- production in proximal tubule cells, likely by inhibiting its translation. Overall preservation of protein synthesis, and comparable MCP-1 suppressions, suggest a semi-selective blockade within the LPS inflammatory mediator cascade. These results, coupled with increases in gut TNF- / MCP-1 mRNAs, imply that gentamicin may exert protean, countervailing, actions on systemic cytokine / chemokine production during Gram negative sepsis.