Cerebrospinal fluid (CSF) concentrations are used as a surrogate measure of central nervous system (CNS) availability of drugs. Systemically administered drugs can reach CSF either directly via passage across the choroid plexus, or indirectly by passage across the blood-brain barrier (BBB) followed by diffusion/convection transport from the interstitial fluid (ISF) to CSF. This review focuses on the physiological and pharmacokinetic variables that must be considered in the interpretation of the CSF-to-plasma concentration gradient. A survey of the literature suggests that the equilibrated CSF-to-unbound plasma concentration ratio reflects the balance between drug permeability across the blood-CNS barriers and the sink action of CSF turnover. As lipophilicity and membrane permeability increase, the CSF-to-plasma unbound concentration ratio rises from well below 1 toward unity. Deviations are noted in that lipophilic drugs highly bound to CSF proteins had ratio exceeding unity, and lipophilic drugs that were efflux transporter substrates exhibited ratios well less than unity. Drug concentration gradients have been observed between drug in the ISF and CSF, the direction of which depends on the CSF sink action and drug permeability/transport across the BBB and choroid plexus. Despite the complexity of CSF pharmacokinetics, for some drugs a rapid kinetic equilibrium exists between the CSF and biophase, such that CSF concentration can serve as a proximate reference for detailed investigation of factors affecting the intrinsic pharmacodynamics of a centrally acting drug. The applicability and limitations of CSF sampling for assessing the CNS availability and concentration-effect relationship of drug candidates with varying physicochemical properties during drug discovery and development is discussed.