Γ-Aminobutyric acid B (GABA_B) receptors are heterodimers composed of two subunits GABA_B(1) and GABA_B(2), the former existing in two isoforms GABA_B(1a) and GABA_B(1b). The contributions of individual receptor subunits and isoforms to GABA_B auto- and heteroreceptor functions were investigated, using release experiments in cortical slice preparations from corresponding knockout mice. Presynaptic GABA_B autoreceptors are located on GABAergic terminals and inhibit GABA release, whereas presynaptic GABA_B heteroreceptors control the release of other neurotransmitters (e.g. glutamate). Neither baclofen nor the selective antagonist CGP55845 at maximally active concentrations affected [³H]GABA release in slices from GABA_B(1)−/− mice. The amount of [³H]GABA released per pulse was unaffected by the stimulation frequency in slices from GABA_B(1)−/− and GABA_B(2)−/− demonstrating a loss of GABA_B autoreceptor function in these knockout animals. The GABA_B receptor agonist baclofen was ineffective in modulating glutamate release in cortical slices from GABA_B(2)−/− mice, showing that heteroreceptor function was abolished as well. Next we investigated knockout mice for the two predominant GABA_B(1) isoforms expressed in brain, GABA_B(1a) and GABA_B(1b). In cortical, hippocampal and striatal slices from both GABA_B(1a)−/− and GABA_B(1b)−/− mice, the frequency dependence of [³H]GABA released per pulse was maintained, suggesting that both isoforms participate or can substitute for each other in GABA_B autoreceptor function. By contrast, the efficacy of baclofen to inhibit glutamate release was substantially reduced in GABA_B(1a)−/−, but essentially unaltered in GABA_B(1b)−/− mice. Our data suggest that functional GABA_B heteroreceptors regulating glutamate release are predominantly, but not exclusively composed of GABA_B(1a) and GABA_B(2) subunits.