gamma-Aminobutyrylcholine (GABAch) has been reported to exist in mammalian brain tissue, but not, as yet, given a specific physiological role in the CNS. In order to investigate further its occurrence and function in the CNS, two new methods have been developed for its isolation and determination at the picomole level. Its isolation has been achieved by ammonium Reineckate precipitation or by cation-exchange followed by HPLC determination of the dansyl and o-phthaldialdehyde derivatives. Using these methods, no free endogenous GABAch (less than 80 pmol/g) was found in rat, guinea pig, cat, pig marmoset, or human brain tissue. No evidence was obtained, either in vitro or in vivo, for the incorporation of [14C]gamma-aminobutyric acid into GABACh. GABACh was hydrolysed at a low rate (maximum of 45 nmol/h/g of brain tissue) after incubation with rat, guinea pig, or cat brain minces and homogenates. These results fail to confirm the data of other investigators, and the possible reasons for this are discussed.