A comprehensive in vivo evaluation of brain penetrability and central nervous system (CNS) pharmacokinetics of atomoxetine in rats was conducted using brain microdialysis. We sought to determine the nature and extent of transport at the blood-brain barrier (BBB) and blood-cerebrospinal fluid barrier (BCB) and to characterize brain extracellular and cellular disposition. The steady-state extracellular fluid (ECF) to plasma unbound (uP) concentration ratio (C(ECF)/C(uP)=0.7) and the cerebrospinal fluid (CSF) to plasma unbound concentration ratio (C(CSF)/C(uP)=1.7) were both near unity, indicating that atomoxetine transport across the BBB and BCB is primarily passive. On the basis of the ratios of whole brain concentration to C(ECF) (C(B)/C(ECF)=170), brain cell (BC) concentration to C(ECF) (C(BC)/C(ECF)=219), and unbound brain cell concentration to C(ECF) (C(uBC)/C(ECF)=2.9), we conclude that whole brain concentration does not represent the concentration in the biophase and atomoxetine primarily partitions into brain cells. The distributional clearance at the BBB (Q(BBB)=0.00110 l/h) was estimated to be 12 times more rapid than that at the BCB (Q(BCB)=0.0000909 l/h) and similar to the clearances across brain parenchyma (CL(ECF-BC)=0.00216 l/h; CL(BC-ECF)=0.000934 l/h). In summary, the first detailed examination using a quantitative microdialysis technique to understand the brain disposition of atomoxetine was conducted. We determined that atomoxetine brain penetration is high, movements across the BBB and BCB occur predominantly by a passive mechanism, and rapid equilibration of ECF and CSF with plasma occurs.