Nociceptin (NOC), an endogenous ligand of the opioid receptor-like 1 receptor, is thought to be involved in learning and memory processes. Since acetylcholine (ACh) is involved in hippocampal function, and the hippocampus plays a critical role on the learning and memory function, hippocampal ACh release in NOC-receptor knockout mice was examined using an in vivo microdialysis method. The release of hippocampal ACh was largely increased in the knockout mice. Furthermore, in the knockout mice, an enhanced hippocampal theta rhythm, which is known to be linked to hippocampal memory function, was also observed. Immunohistochemically, in septum, co-existence of NOC receptor with cholinergic, but not with GABAergic neurons, was verified. The findings demonstrate that the NOC receptor is involved in hippocampal cholinergic function.