In the mammalian nervous system, GABAA receptors exist in an enormous diversity of subtypes. Six , three , three , the , , , , and three subunits can and most of them actually do assemble into a multitude of different homo- or hetero-pentameric receptors, each of them displaying unique pharmacological properties. These receptors are targeted by various clinically relevant drugs such as for example the widely prescribed benzodiazepines, which can exert many different effects, including anxiolytic, sedative, hypnotic, amnesic, anticonvulsant, and muscle relaxant effects. The different benzodiazepine-induced behavioural responses seem to be predominantly mediated by GABAA receptors subtypes containing specific subunits having their effects mostly on the following receptors-configurations: 1n2, 2n2, 3n2 and 5n2. This leads to the search for novel ligands that discriminate between different -containing receptors. Since 5-containing GABAA receptors are located in the hippocampus, 5-selective positive allosteric modulators are expected to have negative effects on memory and cognition, while negative allosteric modulators are thought to improve cognition and memory and might possibly be a future treatment of Alzheimers, schizophrenia, Down-syndrome, autism and other related diseases. Some 5-preferring (selective) compounds have already been identified, such as SH-053-2'F-R-CH3, SH-053-2‘F-R-COOH and MP-III-022. The aim of the current study was to learn more about the molecular determinants and the associated amino acids of the GABAA receptors that lead to 5-selectivity. A sequence alignment of the subunits 1, 2, 3 and 5 indicates that two amino-acids located in the B and C loop of the 5-subunit might contribute to subtype selectivity: P197 in B loop and T239 in C loop. Therefore, wildtype and point mutated subunits have been constructed and expressed in transfected HEK-293 cells: 5(P197T)32, 5(T239S)32, 3(T215P)32, 3(S257T)32, 3(wt)32 und 5(wt)32. The receptors were tested using radioligand binding assays by measuring the displacement of [3H]Ro 15-4513 by the 5 selective compounds SH-053-2'F-R-CH3, SH-053-2‘F-R-COOH and MP-III-022. In this study there was no evidence that the amino acid P197 in the alpha5 subunit in B loop is responsible for the 5-selectivity. However, SH-053-2‘F-R-COOH showed in comparison with the 5- or 3-wildtype a lower affinity to the C loop mutant 5(T239S)32 and higher affinity to C loop mutant 3(S257T). Accordingly, the amino acid 5 T239 in loop C could be a determinant of 5 - binding selectivity.