Up to 60% of gamma-[3H]aminobutyric acid ([3H]GABA) bound specifically to rat cerebellar membranes in the absence of Ca2+ was insensitive to the GABAA antagonist bicuculline and to the GABAB agonist baclofen. This indicates that a significant component of specifically bound [3H]GABA is associated with non-GABAA, non-GABAB binding sites. The presence of this binding component appeared seasonal, peaking in the month of September (early spring) each year over a 4-year period. The calcium independence and bicuculline and baclofen insensitivity of the binding indicate that this binding is not to the classical GABAA and GABAB binding sites. High concentrations of muscimol and isoguvacine inhibited non-GABAA, non-GABAB binding. Scatchard analysis of the non-GABAA, non-GABAB binding sites indicated two kinetic components: KD1 = 42 nM and KD2 = 9.2 microM; Bmax1 = 1.6 pmol/mg of protein and Bmax2 = 28 pmol/mg of protein.