Presynaptic GABAB receptor (GABABR) heterodimers are composed of GB1a/GB2 subunits and critically influence synaptic and cognitive functions. Here, we explored local GABABR activation by integrating optical tools for monitoring receptor conformation and synaptic vesicle release at individual presynaptic boutons of hippocampal neurons. Utilizing fluorescence resonance energy transfer (FRET) spectroscopy, we detected a wide range of FRET values for CFP/YFP-tagged GB1a/GB2 receptors that negatively correlated with release probabilities at single synapses. High FRET of GABABRs associated with low release probability. Notably, pharmacological manipulations that either reduced or increased basal receptor activation decreased intersynapse variability of GB1a/GB2 receptor conformation. Despite variability along axons, presynaptic GABABR tone was dendrite specific, having a greater impact on synapses at highly innervated proximal branches. Prolonged neuronal inactivity reduced basal receptor activation, leading to homeostatic augmentation of release probability. Our findings suggest that local variations in basal GABA concentration are a major determinant of GB1a/GB2 conformational variability, which contributes to heterogeneity of neurotransmitter release at hippocampal synapses.