Glomerular particles were isolated from the bovine cerebellar vermis and studied in vitro to further assess the possibility that gamma-aminobutyric acid (GABA) is utilized as a neurotransmitter in this synaptic complex. Cerebellar glomeruli accumulated [3H]GABA at two different high affinity sites, with affinities (KT) of 2.2 X 10(-6) M and 3.0 X 10(-5) M. These uptake sites could not be distinguished on the basis of their temperature sensitivities, sodium dependence, substrate specificities or responses to metabolic inhibitors. Although an exchange process contributed to the uptake measured in these experiments, a considerable amount of the [3H]GABA accumulated by glomerular particles was stored in an osmotically-sensitive, nonexchangeable pool. Glomerular particles preloaded with [3H]GABA exhibited a Ca2+-independent release of this amino acid in response to membrane depolarization. However, when preloaded glomerular particles were exposed to unlabeled GABA, which presumably displaced [3H]GABA from the exchangeable pool, a K+-evoked and Ca2+-dependent release of the remaining [3H]GABA occurred. The observed net uptake, together with the depolarization-induced and Ca2+-dependent release, of [3H]GABA from glomerular particles supports the suggestion that functionally active GABAergic synapses are present in these structures.