One prime feature of alcoholic liver disease (ALD) is iron accumulation in hepatic macrophages/Kupffer cells (KC) associated with enhanced NF-B activation. Our recent work demonstrates a peroxynitrite-mediated transient rise in intracellular labile iron (ILI response) as novel signaling for endotoxin-induced IB kinase (IKK) and NF-B activation in rodent KC. The present study investigated the mechanism of KC iron accumulation and its effects on ILI response in experimental ALD. We also tested ILI response in human blood monocytes. Chronic alcohol feeding in rats results in increased expression of transferrin (Tf) receptor-1 and hemochromatosis gene (HFE), enhanced iron uptake, an increase in non-heme iron content, and accentuated ILI response for NF-B activation in KC. Ex vivo treatment of these KC with an iron chelator abrogates the increment of iron content, ILI response, and NF-B activation. The ILI response is evident in macrophages derived from human blood monocytes by PMA treatment but not in vehicle-treated monocytes, and this differentiation-associated phenomenon is essential for maximal TNF release. PMA-induced macrophages load iron dextran and enhance ILI response and TNF release. These effects are reproduced in KC selectively loaded in vivo with iron dextran in mice and more importantly aggravate experimental ALD. Our results suggest enhanced iron uptake as a mechanism of KC iron loading in ALD and demonstrate the ILI response as a function acquired by differentiated macrophages in man and as a priming mechanism for ALD.