Hepatic macrophages are sensitized to alcohol in 24 h due to increases in the endotoxin receptor, CD14; however, desensitization to lipopolysaccharide (LPS), which occurred earlier, could not be explained by changes in CD14. Therefore, the purpose of this work was to attempt to understand factors responsible for ethanol-induced desensitization to LPS in hepatic macrophages. Rats were given ethanol (5 g/kg body wt) intragastrically, and hepatic macrophages were isolated 2 h later. After addition of endotoxin, intracellular Ca²⁺ concentration ([Ca²⁺]_i) was measured using fura 2 and tumor necrosis factor (TNF)- was measured by ELISA. Ethanol given 2 h before injection of LPS totally prevented liver injury and blunted LPS-induced increases in [Ca²⁺]_i and TNF- in hepatic macrophages. Furthermore, the protein kinase C (PKC) agonist phorbol 12-myristate 13-acetate and acute ethanol treatment both activated PKC and largely prevented the influx of [Ca²⁺]_i caused by LPS. Sterilization of the gut with antibiotics completely blocked all effects of ethanol on [Ca²⁺]_i and TNF- release. Thus ethanol-induced desensitization of hepatic macrophages correlates with gut-derived endotoxin after ethanol and involves the effect of PKC on voltage-dependent Ca²⁺ channels.