The valid measurement of GABA concentration in the CSF for studying in vivo alterations of central nervous system GABAergic activity requires that consideration be given to several factors that affect GABA concentration. These factors include in vivo variables such as the caudocranial concentration gradient in CSF, age, sex, precursor intake, medications, and physical activity, as well as in vitro alterations that can occur during collection, storage, preparation of sample, and analysis. The influence of these factors can be minimized with proper methodology.