A number of analogues of the low-efficacy partial GABA_A agonist 5-(4-piperidyl)-3-isoxazolol (4-PIOL, 5), in which the 4-position of the 3-isoxazolol ring was substituted by different groups, were synthesized and tested as GABA_A receptor ligands. Substituents of different size and structural flexibility such as alkyl, phenylalkyl, diphenylalkyl, and naphthylalkyl were explored. Pharmacological characterization of the synthesized compounds was carried out using receptor binding assays and by electrophysiological experiments using whole-cell patch-clamp techniques. Whereas none of these compounds significantly affected GABA_B receptor sites or GABA uptake, they did show affinity for the GABA_A receptor site. While alkyl or benzyl substitution, compounds 7a-h, provided receptor affinities comparable with that of 5 (K_i = 9.1 μM), diphenylalkyl and naphthylalkyl substitution, as in compounds 7m-t, resulted in a dramatic increase in affinity relative to 5. The 3,3-diphenylpropyl and the 2-naphthylmethyl analogues, compounds 7s and 7m, respectively, showed the highest affinities of the series (K_i = 0.074 μM and K_i = 0.049 μM). In whole-cell patch-clamp recordings from cultured cerebral cortical neurons, all of the tested compounds were able to inhibit the effect of the specific GABA_A agonist isoguvacine (1), compounds 7m and 7s showing antagonist potency (IC₅₀ = 0.37 μM and IC₅₀ = 0.02 μM) comparable with or markedly higher than that of the standard GABA_A antagonist 4 (IC₅₀ = 0.24 μM). Highly potent convulsant activity was demonstrated in mice with compounds 7m (ED₅₀ = 0.024 μmol/kg) and 7s (ED₅₀ = 0.21 μmol/kg) after intracerebroventricular administration, whereas no effects were found after subcutaneous administration. According to a previously proposed pharmacophore model for GABA_A receptor agonists, a receptor cavity in the vicinity of the 4-position of the 3-isoxazolol ring in 4-PIOL exists. A molecular modeling study, based on compounds 7o, m,l,q,s, was performed to explore the dimensions and other properties of the receptor cavity. This study demonstrates the importance of the arylalkyl substituents in 7m and 7s and the considerable dimensions of this proposed receptor cavity.