We have reported that the microinjection of neostigmine into the hippocampus of rats induced responses similar to stress responses in terms of catecholamines and glucose in plasma. In order to test the hypothesis that hippocampal neostigmine injection is a possible animal model of acute stress responses, we investigated c-Fos expression in the hypothalamic paraventricular nucleus (PVN) and plasma levels of adrenocorticotrophic hormone (ACTH) after hippocampal neostigmine injection and compared these levels with those resulting from stressful conditions such as immobilization and insulin-induced hypoglycemia. The patterns of expression of Fos-ir in the PVN after microinjection of neostigmine into the hippocampus were not different from those seen in the two stressful situations. After microinjection of neostigmine, plasma ACTH levels significantly increased. Taken together, the results of this study indicate that microinjection of neostigmine into the hippocampus is a potential experimental model for acute stress responses.