Ginkgolides are potent blockers of the glycine receptor Cl- channel (GlyR) pore. We sought to identify their binding sites by comparing the effects of ginkgolides A, B and C and bilobalide on alpha1, alpha2, alpha1beta and alpha2beta GlyRs. Bilobalide sensitivity was drastically reduced by incorporation of the beta subunit. In contrast, the sensitivities to ginkgolides B and C were enhanced by beta subunit expression. However, ginkgolide A sensitivity was increased in the alpha2beta GlyR relative to the alpha2 GlyR but not in the alpha1beta GlyR relative to the alpha1 GlyR. We hypothesised that the subunit-specific differences were mediated by residue differences at the second transmembrane domain 2' and 6' pore-lining positions. The increased ginkgolide A sensitivity of the alpha2beta GlyR was transferred to the alpha1beta GlyR by the G2'A (alpha1 to alpha2 subunit) substitution. In addition, the alpha1 subunit T6'F mutation abolished inhibition by all ginkgolides. As the ginkgolides share closely related structures, their molecular interactions with pore-lining residues were amenable to mutant cycle analysis. This identified an interaction between the variable R2 position of the ginkgolides and the 2' residues of both alpha1 and beta subunits. These findings provide strong evidence for ginkgolides binding at the 2' pore-lining position.