The time course of nicotine metabolite appearance in brain from 5 min-18 hr after subcutaneous administration of S-(-)-[3H-N-methyl]nicotine was determined. Results demonstrated that metabolite appearance in brain was greatest at 4 hr postadministration, whereas levels of nicotine were greatly diminished at this time point. For determination of N-demethylated metabolites, (+/-)-[2'-14C]nicotine was administered subcutaneously to rats, and the presence of nicotine and nicotine metabolites in brain supernatant was determined 4 hr postadministration. Using high-performance liquid radiochromatographic analysis, nicotine and three nicotine metabolites (cotinine, nornicotine, and norcotinine) were identified in brain, together with a fourth minor, unidentified metabolite. After subcutaneous administration of S-(-)-[G-3H]cotinine, significant amounts of cotinine were found in brain over an 18-hr postadministration period; however, no cotinine metabolites were detected. Therefore, cotinine is able to pass the blood-brain barrier and access the central nervous system, but is not biotransformed in brain. Thus, this is the first report of norcotinine as a central nervous system nicotine metabolite. Data indicate that norcotinine detected in brain after peripheral nicotine administration most likely originates from 5'-C-oxidation of brain nornicotine, rather than from N-demethylation of cotinine, as occurs peripherally. Because peripheral biotransformation of nicotine to nornicotine is a minor pathway, the relatively high levels of nornicotine found in brain after peripheral nicotine administration suggest that nornicotine is formed via oxidative N-demethylation of nicotine locally in brain. Nornicotine is pharmacologically active; thus, its presence in brain after peripheral nicotine administration indicates that nornicotine may contribute to the neuropharmacological effects of nicotine and tobacco use.