The relationship between the serum imipramine concentration and its antidepressant effects remain undefined despite > 30 years of clinical investigation. No study to date has assessed the kinetic relationships between the concentrations of imipramine and its metabolites in plasma and in various brain structures. In this study, we examine the pharmacokinetics of imipramine (IMI) and its desmethylated and hydroxylated metabolites in rats given IMI chronically (20 mg/kg, intraperitoneally twice a day for 14 days). The concentrations in serum, cerebrospinal fluid, and six brain structures were measured by high-performance liquid chromatography at 13 different times from 0.5 to 120 h after the end of treatment. The concentrations of IMI, desipramine (DMI), and didesmethylimipramine (DDMI) in brain tissue were much higher than in the serum; concentrations were maximal at 1-2 h in the serum and the brain, which is indicative of the rapid metabolism of IMI with immediate and massive entry of the metabolites into the brain. The elimination halflives of desmethylated compounds increased with the degree of desmethylation, and DDMI was still present in brain tissue 96 h after the end of treatment. These results suggest that DDMI should be taken into account in clinical investigations of the effects of serum concentrations of IMI. The hydroxylated metabolites 2-OH imipramine (2-OH IMI) and 2-OH desipramine (2-OH DMI) were detected in serum, but not in cerebral tissue. The 10-OH metabolites were detected in both serum and brain, but the antidepressant action of these metabolites have not been clearly established. Finally, there were significant differences in the distributions of IMI and several of its metabolites in brain structures. Such differences may have clinical relevance if they also occur in humans.