New experimental methodologies were applied to measure the unbound brain-to-plasma concentration ratio (K(p,uu,brain)) and the unbound CSF-to-plasma concentration ratio (K(p,uu,CSF)) in rats for 43 structurally diverse drugs. The relationship between chemical structure and K(p,uu,brain) was dominated by hydrogen bonding. Contrary to popular understanding based on the total brain-to-plasma concentration ratio (logBB), lipophilicity was not a determinant of unbound brain exposure. Although changing the number of hydrogen bond acceptors is a useful design strategy for optimizing K(p,uu,brain), future improvement of in silico prediction models is dependent on the accommodation of active drug transport. The structure-brain exposure relationships found in the rat also hold for humans, since the rank order of the drugs was similar for human and rat K(p,uu,CSF). This cross-species comparison was supported by K(p,uu,CSF) being within 3-fold of K(p,uu,brain) in the rat for 33 of 39 drugs. It was, however, also observed that K(p,uu,CSF) overpredicts K(p,uu,brain) for highly effluxed drugs, indicating lower efflux capacity of the blood-cerebrospinal fluid barrier compared to the blood-brain barrier.