The inhibitory action of bicyclic isoxazole gamma-aminobutyric acid (GABA) analogues and their 4,4-diphenyl-3-butenyl (DPB) substituted derivatives has been investigated in cortical neurones and astrocytes as well as in human embryonic kidney (HEK 293) cells transiently expressing either mouse GABA transporter-1 (GAT-1), GAT-2, -3 or -4. It was found that 4,5,6,7-tetrahydroisoxazolo(4,5-c)pyridin-3-ol (THPO) and 5,6,7,8-tetrahydro-4H-isoxazolo[4,5-c]azepin-3-ol (THAO) displayed some inhibitory activity on GAT-1 and GAT-2, where the compounds exhibited a slightly lower potency on GAT-2 compared to GAT-1. DPB substituted THPO displayed higher inhibitory potency than the parent compound regarding the ability to inhibit GABA uptake via GAT-1 and GAT-2. Concerning the inhibitory mechanism, THPO, THAO and DPB-THPO were competitive inhibitors on GAT-1 transfected HEK 293 cells and the same mechanism was observed for THPO in GAT-3 transfected cells. Regarding GABA uptake into neurones and astroglia cells THAO and DPB-THAO both displayed competitive inhibitory action. The observations that THPO, THAO as well as their DPB derivatives act as competitive inhibitors together with earlier findings such as potent anticonvulsant activity, lack of proconvulsant activity and the ability of THPO to increase extracellular GABA concentration, indicate that these bicyclic isoxazole GABA analogues and their DPB derivatives may be useful lead structures in future search for new antiepileptic drugs.