Agmatine, decarboxylated arginine, is an NMDA receptor (NMDAr) antagonist, nitric oxide synthase (NOS) inhibitor and putative neuromodulator. NMDAr and NOS inhibitors are known to prevent opioid tolerance and self-administration; it has been previously reported that the development of morphine tolerance and fentanyl self-administration is reduced by systemic agmatine injection. However, it is assumed that the site(s) of agmatine action is in the central nervous system. Our group previously identified a CNS site of action by showing that intrathecally administered agmatine prevents morphine spinal tolerance and well as hyperalgesia in neuropathic and inflammatory pain models. In the present study, we evaluated the pharmacology and pharmacokinetics of intracerebroventricular (i.c.v.) agmatine in mice made tolerant to supraspinally delivered morphine (1) and mice self-administering oral fentanyl (2). In the first experiment (1), ICR mice were pretreated with i.c.v. morphine (10 nmol, b.i.d. for 3 days). On day 4, the analgesic response (tail flick assay) to a probe dose of i.c.v. morphine was assessed. In the second experiment (2) ICR mice were trained to bar press for orally delivered fentanyl (70 µL) in daily 2 hour operant sessions. Agmatine treatment (10 nmol, i.c.v.) given once every other day completely prevented the development of either morphine tolerance or fentanyl self-administration. Brain pharmacokinetics were measured following i.c.v. injections of agmatine (120 nmol) and whole brains were analyzed for agmatine content using HPLC. Agmatine levels in whole brain were detected for more than a day following i.c.v. agmatine delivery. Agmatine given i.c.v. is measurable in brain and effective in preventing development of morphine tolerance and fentanyl self-administration for days between treatments.