THE GABA receptor ρ subunits are thought to form bicuculline-insensitive and picrotoxinin-sensitive GABAc receptors. We have investigated the role of the amino acid at position 309 in transmembrane segment M2 of the human ρ1 subunit as a determinant for picrotoxinin sensitivity. The mutant ρ1P309S was constructed by exchanging proline 309 for serine, the corresponding amino acid of the human ρ2 subunit. Whole-cell recordings from HEK-293 cells transfected with ρlP309S cDNA revealed that the sensitivity of the ρlP309S channels for picrotoxinin was four-fold lower than that of the wild type ρ1 subunit. The affinity of the mutant receptor for GABA was only slightly changed. These results provide direct evidence that the amino acid at position 309 is an important determinant for the picrotoxinin sensitivity of GABA receptors formed by the ρ subunits.