Abstract: Previously, we have shown a significant increase in number of GABAB receptor binding sites in neocortex and thalamus of lethargic (lh/lh) mice, a mutant strain exhibiting absence seizures. This study was performed to test our hypothesis that presynaptic GABAB receptors would inhibit [3H]GABA release to a greater degree in lh/lh mice compared with their nonepileptic littermates (designated +/+). Synaptosomes isolated from neocortex and thalamus of age‐matched male lh/lh and +/+ mice were similar in uptake of [3H]GABA. In the neocortical preparation, baclofen dose‐dependently inhibited [3H]GABA release evoked by 12 mM KCl, an effect mediated by GABAB receptors. The maximal inhibition (Imax) value was significantly greater (80%) in lh/lh than +/+ mice, whereas the IC50 (3 µM) was unchanged. In the thalamic preparation, the effect of baclofen (50 µM) was 58% less robust in lh/lh mice. Other effects mediated by GABAB receptors (inhibitions in Ca2+ uptake and cyclic AMP formation) were also significantly reduced in thalamic synaptosomes from lh/lh mice. These data suggest a greater presynaptic GABAB receptor‐mediated effect in neocortex and a reduced effect in thalamic nuclei of lh/lh mice. It is possible that selective effects of presynaptic GABAB receptors on GABA release in neocortex and thalamic nuclei of lh/lh mice may contribute to mechanisms underlying absence seizures.