The inhibitory neurotransmitter γ-aminobutyric acid (GABA) acts through various types of receptors in the central nervous system. GABA_ρ receptors, defined by their characteristic pharmacology and presence of ρ subunits in the channel structure, are poorly understood and their role in the cortex is ill-defined. Here, we used a targeted pharmacological, NMR-based functional metabolomic approach in Guinea pig brain cortical tissue slices to identify a distinct role for these receptors. We compared metabolic fingerprints generated by a range of ligands active at GABA_ρ and included these in a principal components analysis with a library of other metabolic fingerprints obtained using ligands active at GABA_A and GABA_B, with inhibitors of GABA uptake and with compounds acting to inhibit enzymes active in the GABAergic system. This enabled us to generate a metabolic “footprint” of the GABAergic system which revealed classes of metabolic activity associated with GABA_ρ which are distinct from other GABA receptors. Antagonised GABA_ρ produce large metabolic effects at extrasynaptic sites suggesting they may be involved in tonic inhibition.