A series of novel 5-(4-piperidyl)-3-isoxazolol (4-PIOL) analogues where the 4-position of the 3-isoxazolol ring was substituted with groups of different size, flexibility, and lipophilicity have been characterised. Their activity as agonists and/or antagonists on human alpha(1)beta(2)gamma(2S) GABA(A) receptors expressed in Xenopus oocytes was studied using two-electrode voltage clamp electrophysiology. Methyl- and ethyl-substituted 4-PIOL analogues were characterised as partial agonists since weak agonist responses could be potentiated with lorazepam and inhibited by the competitive antagonist 2-(3-carboxypropyl)-3-amino-6-methoxyphenyl-pyradizinum bromide (SR95531). All larger substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL converted the compounds into pure competitive antagonists. Additionally, for GABA, 4,5,6,7-tetrahydroisoxazolo[5,4-c]pyridin-3-ol (THIP), piperidine-4-sulphonic acid (P4S), and 5-(4-piperidyl)-3-isothiazolol (thio-4-PIOL), a negative linear correlation was found between the agonist efficacy of the compound and the ability of lorazepam to potentiate EC(95) responses. Furthermore, a positive linear correlation between the lipophilicity of the substituents in the 4-position of the 3-isoxazolol ring of 4-PIOL and the antagonist affinity was found. These data suggest that the GABA(A) receptor contains a hydrophobic binding pocket at the GABA recognition site and that the binding of the 4-PIOL analogues is largely determined by the transfer from the aqueous phase to the hydrophobic pocket.