The α4βδ subtype of the γ-aminobutyric acid (GABA) type A receptors (GABAARs) has been shown to be implicated in high-affinity binding of the neuromodulator γ-hydroxybutyric acid (GHB), but may not be the only GHB high-affinity binding sites. Monastrol has been identified as a modulator of GHB high-affinity binding and is furthermore reported as an allosteric modulator selective for the α1β2δ GABAARs. Therefore, structural determinants for selectivity at the two targets were investigated. 39 structural diverse monastrol analogues were synthesized by employing the Biginelli cyclocondensation and examined for modulation of GHB high-affinity binding using the GHB-specific ligand [(3)H]NCS-382 [(E,RS)-6,7,8,9-tetrahydro-5-hydroxy-5H-benzocyclohept-6-ylidene)acetic acid] in rat brain homogenate. Only limited modifications were allowed on the monastrol scaffold in order to maintain modulation of GHB high-affinity binding. However, three analogues of monastrol (11, 12 and 24) enhanced the maximal binding of [(3)H]NCS-382 to a higher maximal level than seen for monastrol itself. Selected compounds were further characterized as modulators at α1β2δ, α1β2γ2s and α1β2 GABAARs. Most of these modulators were shown to have δ-specific GABA-potentiating effects. The dual effect shown for monastrol to modulate the GHB high-affinity binding and α1β2δ GABAAR activity was also shown for the compounds 11, 18 and 24. Compound 29 displayed minimal modulatory effect on GABAARs and therefore appears to be a GHB high-affinity binding preferring modulator. However, compounds 34 and 37 were shown to be α1β2δ GABAAR selective modulators, without modulatory effects on GHB high-affinity binding. Thus, our study shows that minor modifications in the structure of monastrol affects the selectivity profile for the two targets under study enabling separation of the dual activity.