An additional administration of high dose ethanol to chronic alcohol-fed rats led to a decrease in endotoxin clearance and an increase in endotoxin accumulation in the spleen accompanied by an elevation of tumour necrosis factor (TNF) levels in the portal vein. Endotoxin uptake and TNF production by Kupffer cells (KC) and splenic macrophages in the chronic ethanol load rats were significantly greater than those in the control rats. When these cells were precultured in the medium containing 10 to 100 mmol/L ethanol, the endotoxin uptake and TNF production of KC were decreased. However, this did not affect the endotoxin uptake and TNF production of splenic macrophages. The hepatic production of endotoxin binding protein was increased when KC were preincubated in the medium containing ethanol and the resultant culture supernatant was added to the hepatocyte culture system. This endotoxin binding protein was proved to enhance the uptake of endotoxin and suppressed the production of TNF in the KC. When KC and hepatocytes were isolated from chronically alcohol-fed rats, further addition of ethanol to the culture medium of KC did not affect the hepatic production of endotoxin binding protein. The increase in hepatic production of endotoxin binding protein may serve as a defence mechanism against endotoxicity. There is a possibility that an impairment of this defence mechanism has a pivotal role in the development of endotoxaemia and endotoxicity in chronic alcoholics.