Serial biochemical studies of a rat cortical tissue culture system in which synapses regularly form showed that gamma-aminobutyric acid (GABA) is present in the cultures and increases with their maturation. The tissue GABA concentration in mature cultures is similar to that of adult rat cortex in vivo. The synthetic enzyme, glutamate decarboxylase, also increases with age as does high affinity GABA uptake. GABA uptake was blocked by L-2,4-diaminobutyrate (DABA) and had the properties of neuronal GABA uptake. Specific release by depolarizing media of both exogenous [3H]GABA and GABA synthesized from D-[U-14C]glucose was demonstrated. The GABA released by high potassium media had higher specific activity and a greater contribution from glucose (as compared to acetate) than GABA found in the medium in the absence of depolarization. Calcium dependency of evoked GABA release could be shown only after pretreatment of cultures with ethyleneglycol-bis-(beta-aminoethyl ether)-N,N'-tetraacetic acid or EGTA. Synaptosomes may exhibit greater calcium dependence of evoked transmitter release than intact cells in culture because their intracellular calcium stores are depleted during preparation. Glycine uptake by the cultures was much less in amount than was GABA uptake, and specific release of glycine could not be demonstrated. Specific binding of both a GABA agonist ([3H]muscimol) and an antagonist ([3H]bicuculline) was shown by membranes prepared from the cultures. By contrast, when [3H]muscimol binding to intact cells was studied, essentially all binding was sodium dependent and had the properties of GABA uptake binding. We conclude that the use of [3H]muscimol for receptor studies is valid only after the elimination of GABA uptake systems. Biochemical data from these studies support the concept that GABA is the transmitter for many cortical synapses. Glycine and taurine are not likely to be transmitters in these cortical cultures. When considered together with physiological data from the preceding paper, we have satisfied Werman's criteria (see ref. 36) for accepting GABA as the major inhibitory transmitter in the cortical culture system.