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To study endotoxin tolerance in the subarachnoid space 0.1 mg of endotoxin derived from Neisseria meningitidis was injected intracisternally into rabbits on 2 consecutive days. On day 1 the maximum peak level of TNFα was 7 ng/ml 2 h after injection, whereas on day 2 the highest levels were 3.6 ng/ml and 3.7 ng/ml, respectively, 1 and 2 h after injection. Pretreatment with intravenous endotoxin 5 or 21 h before consecutive intracisternal endotoxin did not affect the cerebrospinal fluid (CSF) levels of TNFα. In contrast, there was a marked endotoxin tolerance with respect to TNFα in the systemic circulation. Cells appearing in the CSF 5, 12 and 20 h after intracisternal injection of endotoxin were harvested, cultured, and then stimulated with 0.1 mg/ml of endotoxin. In 10 experiments a marked TNFα production in the range 10–70 ng/ml was detected in the supernatants, whereas unstimulated cells did not produce TNFα. We conclude that tolerance to endotoxin does not develop in the subarachnoid space as evaluated by the present experimental design. The pattern of TNFα production and endotoxin tolerance is distinctly different in the subarachnoid space and systemic circulation.