Changes in the levels of guanidino compounds in the cortex, hippocampus, striatum, hypothalamus, midbrain, pons and medulla oblongata and cerebellum were analyzed by high performance liquid chromatography after an injection of ferric chloride into the sensory motor cortex of SD rats. Levels of guanidinoacetic acid (GAA) and methylguanidine (MG) changed greatly 15 and 30 min after the injection, but recovered to normal levels 24 to 48 hours after the injection. GAA and MG increased two or three times the normal level 2 months after the injection, at which time iron-induced epileptogenic foci were formed. Levels of other guanidino compounds, i.e., N-acetylarginine, guanidinoacetic acid, arginine (Arg), homoarginine and creatinine (CRN) also changed, though the extent of the changes was not as marked as with GAA and MG. Rapidly increased generation of hydroxyl radical and peroxide intermediate radical(s) was observed by electron spin resonance analysis after addition of ferric chloride and hydrogen peroxide to rat brain homogenate. Levels of MG and GAA also increased. A significant relationship between the generation of hydroxyl radical and the formation of MG was recognized. The formation of MG in the system was dependent on the concentration of CRN but independent of the concentration of Arg and GAA. These results suggest that MG formed from CRN may act on neurones after iron injection into the rat brain, thus forming epileptogenic feci.