In vivo microdialysis was used to assess the effects of Novelty, persistent pain (Formalin test) and stress (Restraint) on hippocampal acetylcholine (ACh) release. Experiments were carried out during the dark phase, i.e. during the active period of the animal, and consisted of four experimental phases: Baseline (30 min), Novelty (30 min), Formalin test (90 min) and Restraint (30 min); each animal was consecutively exposed to all phases. The extracellular levels of ACh in the dorsal hippocampus were estimated by measurement of its concentration in the perfusion fluid by high-performance liquid chromatography with electrochemical detection. The introduction to a new environment (Novelty) induced in all rats higher ACh levels than Baseline. Formalin treatment decreased ACh release only in animals considered 'Inactive' during the Novelty phase while no modification in ACh release was observed in the 'Active' ones. Restraint did not produce any modification of ACh release but increased Corticosterone plasma levels both in sham- and formalin-treated animals. Results indicate that Novelty, but not Formalin or Restraint, increases ACh release in the hippocampus and that the type of behavioral state displayed by the animal at the time of formalin injection determines the response of the septo-hippocampal cholinergic pathway.